Card-domain containing polypeptides, encoding nucleic acids, and methods of use

ABSTRACT

The invention provides caspase recruitment domain (CARD)-containing polypeptides and functional fragments thereof, encoding nucleic acid molecules, and specific antibodies. Also provided are screening methods for identifying CARD-associated polypeptides (CAPs), and for identifying agents that alter the association of a CARD-containing polypeptide with itself or with a CAP. Further provided are methods of altering a biochemical process modulated by a CARD-containing polypeptide, and methods of diagnosing a pathology characterized by an increased or decreased level of a CARD-containing polypeptide.

This application claims the benefit of U.S. Provisional Application No.60/257,457, filed Dec. 21, 2000, which is incorporated herein byreference.

This invention was made with United States Government support undergrant number DBI-0078731 awarded by the National Science Foundation. TheU.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the fields of molecular biology andmolecular medicine and more specifically to the identification ofproteins involved in programmed cell death, cytokine processing andreceptor signal transduction, and associations of these proteins.

2. Background Information

Programmed cell death is a physiologic process that ensures homeostasisis maintained between cell production and cell turnover in essentiallyall self-renewing tissues. In many cases, characteristic morphologicalchanges, termed “apoptosis,” occur in a dying cell. Since similarchanges occur in different types of dying cells, cell death appears toproceed through a common pathway in different cell types.

In addition to maintaining tissue homeostasis, apoptosis also occurs inresponse to a variety of external stimuli, including growth factordeprivation, alterations in calcium levels, free-radicals, cytotoxiclymphokines, infection by some viruses, radiation and mostchemotherapeutic agents. Thus, apoptosis is an inducible event thatlikely is subject to similar mechanisms of regulation as occur, forexample, in a metabolic pathway. In this regard, dysregulation ofapoptosis also can occur and is observed, for example, in some types ofcancer cells, which survive for a longer time than corresponding normalcells, and in neurodegenerative diseases where neurons die prematurely.In viral infections, induction of apoptosis can figure prominently inthe pathophysiology of the disease process, because immune-based foreradication of viral infections depend on elimination of virus-producinghost cells by immune cell attack resulting in apoptosis.

Some of the proteins involved in programmed cell death have beenidentified and associations among some of these proteins have beendescribed. However, additional apoptosis regulating proteins remain tobe found and the mechanisms by which these proteins mediate theiractivity remains to be elucidated. The identification of the proteinsinvolved in cell death and an understanding of the associations betweenthese proteins can provide a means for manipulating the process ofapoptosis in a cell and, therefore, selectively regulating the relativelifespan of a cell or its relative resistance to cell death stimuli.

The principal effectors of apoptosis are a family of intracellularproteases known as Caspases, representing an abbreviation for CysteineAspartyl Proteases. Caspases are found as inactive zymogens inessentially all animal cells. During apoptosis, the caspases areactivated by proteolytic processing at specific aspartic acid residues,resulting in the production of subunits that assemble into an activeprotease typically consisting of a heterotetramer containing two largeand two small subunits. The phenomenon of apoptosis is produced directlyor indirectly by the activation of caspases in cells, resulting in theproteolytic cleavage of specific substrate proteins. Moreover, in manycases, caspases can cleave and activate themselves and each other,creating cascades of protease activation and mechanisms for“auto”-activation. Thus, knowledge about the proteins that interact withand regulate caspases is important for devising strategies formanipulating cell life and death in therapeutically useful ways. Inaddition, because caspases can also participate in cytokine activationand other processes, knowledge about the proteins that interact withcaspases can be important for manipulating immune responses and otherbiochemical processes in useful ways.

One of the mechanisms for regulating caspase activation involvesprotein-protein interactions mediated by a family of protein domainsknown as caspase recruitment domains (CARDs). The identification ofproteins that contain CARD domains and the elucidation of the proteinswith which they interact, therefore, can form the basis for strategiesdesigned to alter apoptosis, cytokine production, cytokine receptorsignaling, and other cellular processes.

Thus, a need exists to identify proteins that contain CARD domains. Thepresent invention satisfies this need and provides additional advantagesas well.

SUMMARY OF THE INVENTION

In accordance with the present invention, there are providedCARD-containing polypeptides, and functional fragments thereof. Theinvention also provides nucleic acid molecules encoding CARD-containingpolypeptides and active fragments thereof, vectors containing thesenucleic acid molecules and host cells containing the vectors. Theinvention also provides antibodies that can specifically bind toCARD-containing polypeptides, and active fragments thereof.

The present invention also provides a screening assay useful foridentifying CARD-associated polypeptides (CAPs), and for identifyingagents that can effectively alter the association of a CARD-containingpolypeptide with itself or with other proteins. By altering theself-association of a CARD-containing polypeptide or by altering itsinteraction with other proteins, an effective agent may increase ordecrease the level of caspase proteolytic activity or apoptosis in acell.

The invention also provides methods of altering a biochemical processmodulated by a CARD-containing polypeptide, by introducing into the celland expressing a nucleic acid sequence encoding the polypeptide, or anantisense nucleotide sequence that is complementary to a portion of anucleic acid molecule encoding the CARD-containing polypeptide. Suchbiochemical processes include apoptosis, anoikis, cytoskeletalintegrity, NF-κB induction, cytokine processing, cytokine receptorsignaling, and caspase-mediated proteolysis.

The invention also provides methods for diagnosing or prognosing apathology characterized by an increased or decreased level of aCARD-containing polypeptide in a cell, by contacting the test samplewith an agent that can specifically bind a CARD-containing polypeptideor a nucleotide sequence, and determining the amount of specific bindingin the test sample compared to a reference sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence (SEQ ID NO:1) and predicted aminoacid sequence (SEQ ID NO:2) of CARD-10X. The CARD domain nucleotidesequence (SEQ ID NO:3) and amino acid sequence (SEQ ID NO:4), and theFilament domain nucleotide sequence (SEQ ID NO:5) and amino acidsequence (SEQ ID NO:6), are indicated and underlined.

FIG. 2 shows the nucleotide sequence (SEQ ID NO:7) and predicted aminoacid sequence (SEQ ID NO:8) of CARD-11X. The CARD domain nucleotidesequence (SEQ ID NO:9) and amino acid sequence (SEQ ID NO:10), the ERM(ezrin) domain nucleotide sequence (SEQ ID NO:11) and amino acidsequence (SEQ ID NO:12), and the PDZ domain nucleotide sequence (SEQ IDNO:13) and amino acid sequence (SEQ ID NO:14), are indicated andunderlined.

FIG. 3 shows the nucleotide sequence (SEQ ID NO:15) and predicted aminoacid sequence (SEQ ID NO:16) of CARD-12X. The CARD domain nucleotidesequence (SEQ ID NO:17) and amino acid sequence (SEQ ID NO:18) isindicated and underlined.

FIG. 4 shows the nucleotide sequence (SEQ ID:19) and predicted aminoacid sequence (SEQ ID NO:20) of GI 10436238.

FIG. 5 shows the nucleotide sequences of CARD-10X ESTs: GI 9094656 (SEQID NO:21), GI 7132200 (SEQ ID NO:22) and GI 5884878 (SEQ ID NO:23).

FIG. 6 shows the nucleotide sequences of CARD-11X ESTs: GI 6926669 (SEQID NO:24), GI 6143407 (SEQ ID NO:25), GI 2785620 (SEQ ID NO:26), GI1838222 (SEQ ID NO:27), GI 6927709 (SEQ ID NO:28), GI 9720543 (SEQ IDNO:29), GI 9142863 (SEQ ID NO:30), GI 1761194 (SEQ ID NO:31), GI 8151878(SEQ ID NO:32), GI 2007639 (SEQ ID NO:33), GI 8042493 (SEQ ID NO:34), GI2079290 (SEQ ID NO:35), and GI 7044777 (SEQ ID NO:36).

FIG. 7 shows the nucleotide sequence of a CARD-12X EST: GI 10316320 (SEQID NO:37).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel polypeptides involved in programmedcell death, or apoptosis. The principal effectors of apoptosis are afamily of intracellular cysteine aspartyl proteases, known as caspases.Caspase activity in the cell is regulated by protein-proteininteractions. Similarly, protein-protein interactions influence theactivity of other proteins involved in apoptosis. Several proteininteraction domains have been implicated in interactions among someapoptosis-regulating proteins. Among these is the caspase recruitmentdomain, or CARD-containing polypeptide which are so named for theability of the CARD-containing polypeptides to bind caspases. Inaddition to their ability to bind caspases, numerous CARD-containingpolypeptides bind other proteins, including other CARD-containingpolypeptides. Further, CARD-containing polypeptides influence a varietyof cellular and biochemical processes beyond apoptosis, including celladhesion, inflammation and cytokine receptor signaling.

In accordance with the present invention, there are provided isolatedCARD-containing polypeptides or functional fragments thereof.

The term “CARD-containing polypeptide” as used herein refers to aprotein or polypeptide containing a CARD domain. As used herein, theterm “CARD domain” refers to a Caspase Recruitment Domain. A CARD domainis a well known protein domain of approximately 80 amino acids withcharacteristic sequence conservation as described, for example, inHofmann et al., Trends Biochem. Sci. 22:155-156 (1997). CARD domainshave been found in some members of the Caspase family of cell deathproteases. Caspases-1, 2, 4, 5, 9, and 11 contain CARD domains neartheir NH2-termini. These CARD domains mediate interactions of thezymogen inactive forms of caspases with other proteins which can eitheractivate or inhibit the activation of these enzymes.

For example, the CARD domain of pro-caspase-9 binds to the CARD domainof a caspase-activating protein called Apaf-1 (Apoptosis ProteaseActivating Factor-1). Similarly, the CARD domain of pro-caspase-1permits interactions with another CARD protein known as Cardiac (alsoreferred to as RIP2 and RICK), which results in activation of thecaspase-1 protease (Thome et al., Curr. Biol. 16:885-888 (1998)).Furthermore, pro-caspase-2 binds to the CARD protein Raidd (also know asCradd), which permits recruitment of pro-caspase-2 to Tumor NecrosisFactor (TNF) Receptor complexes and which results in activation of thecaspase-2 protease (Ahmad et al., Cancer Res. 57:615-619 (1997)). CARDdomains can also participate in homotypic interactions with themselves,resulting in self-association of polypeptides that contain theseprotein-interaction domains and producing dimeric or possibly evenoligomeric complexes.

CARD domains can be found in association with other types of functionaldomains within a single polypeptide, thus providing a mechanism forbringing a functional domain into close proximity or contact with atarget protein via CARD:CARD associations involving two CARD-containingpolypeptides. For example, the Caenorhabiditis elegans cell death geneced-4 encodes a protein that contains a CARD domain and a ATP-bindingoligomerization domain called an NB-ARC domain (van der Biezen andJones, Curr. Biol. 8:R226-R227). The CARD domain of the CED-4 proteininteracts with the CARD domain of a pro-caspase called CED-3. The NB-ARCdomain allows CED-4 to self-associate, thereby forming an oligomericcomplex which brings associated pro-CED-3 molecules into close proximityto each other. Because most pro-caspases possess at least a small amountof protease activity even in their unprocessed form, the assembly of acomplex that brings the proforms of caspase into juxtaposition canresult in trans-processing of zymogens, producing the proteolyticallyprocessed and active caspase. Thus, CED-4 employs a CARD domain forbinding a pro-caspase and an NB-ARC domain for self-oligomerization,resulting in caspase clustering, proteolytic processing and activation.

In addition to their role in caspase activation, CARD domains have beenimplicated in other cellular processes. Some CARD-containingpolypeptides, for example, induce activation of the transcription factorNF-κB. NF-κB activation is induced by many cytokines and plays animportant role in cytokine receptor signal transduction mechanisms(DiDonato et al., Nature 388:548-554 (1997)). Moreover, CARD domains arefound in some proteins that inhibit rather than activate caspases, suchas the IAP (Inhibitor of Apoptosis Protein) family members, cIAP1 andcIAP2 (Rothe et al., Cell 83:1243-1252 (1995)) and oncogenic mutants ofthe Bcl-10 protein (Willis et al., Cell 96:35-45 (1999)). Also, thoughcaspase activation resulting from CARD domain interactions is ofteninvolved in inducing apoptosis, other caspases are primarily involved inproteolytic processing and activation of inflammatory cytokines (such aspro-IL-1β and pro-IL-18). Thus, CARD-containing polypeptides can also beinvolved in cytokine receptor signaling and cytokine production, and,therefore, can be involved in regulation of immune and inflammatoryresponses.

In view of the function of the CARD domain within the inventionCARD-containing polypeptides or functional fragments thereof,polypeptides of the invention are contemplated herein for use in methodsto alter biochemical processes such as apoptosis, NF-κB induction,cytokine processing, cytokine receptor signaling, and caspase-mediatedproteolysis, thus having modulating effects on cell life and death(i.e., apoptosis), inflammation, cell adhesion, and other cellular andbiochemical processes.

It is also contemplated herein that invention CARD-containingpolypeptides can associate with other CARD-containing polypeptides toform invention hetero-oligomers or homo-oligomers, such as heterodimersor homodimers. In particular, the association of the CARD domain ofinvention polypeptides with another CARD-containing polypeptide, such asApaf-1, CED-4, caspases-1, 2, 9, 11, cIAPs-1 and 2, CARDIAK, Raidd,Dark, CARD4, an invention CARD-containing polypeptide, and the like,including homo-oligomerization, is sufficiently specific such that thebound complex can form in vivo in a cell or in vitro under suitableconditions. Similarly therefore, an invention CARD-containingpolypeptide can associate with another CARD-containing polypeptide byCARD:CARD form invention hetero-oligomers or homo-oligomers, such asheterodimers or homodimers.

In accordance with the present invention, sequences for CARD-containingpolypeptides have been determined. Thus, the present invention providesCARD-containing polypeptides, including the newly identifiedCARD-containing polypeptides designated CARD-10X (SEQ ID NO:2), CARD-11X(SEQ ID NO:8) and CARD-12X (SEQ ID NO:16), and functional fragmentsthereof.

CARD-10X was identified as an unannotated protein product in the NRprotein sequence database (GI 10436238; SEQ ID NO:20, encoded by GI10436237; SEQ ID NO:19). A human EST (GI 9094656; SEQ ID NO:21) and twoESTs for Gallus homologs (GI 7132200; SEQ ID NO:22 and GI 5884878; SEQID NO:23) from CARD-10X have also been identified.

CARD-11X was identified from the nucleotide database of High ThroughputGenomic Sequences (NTGS) (GI 10198542, 9887755) and also from the NRnucleotide database, GI 9665194). Several human ESTs from CARD-11X havebeen identified: GI 6926669 (SEQ ID NO:24), GI 6143407 (SEQ ID NO:25),GI 2785620 (SEQ ID NO:26), GI 1838222 (SEQ ID NO:27), GI 6927709 (SEQ IDNO:28), GI 9720543 (SEQ ID NO:29), GI 9142863 (SEQ ID NO:30), GI 1761194(SEQ ID NO:31), GI 8151878 (SEQ ID NO:32), GI 2007639 (SEQ ID NO:33), GI8042493 (SEQ ID NO:34), GI 2079290 (SEQ ID NO:35), and GI 7044777 (SEQID NO:36).

CARD-12X was identified from the database of HTGS (GI 8224622). A humanEST from CARD-12X has also been identified: GI 10316320 (SEQ ID NO:37).

The invention CARD-10X, -11X and -12X nucleic acid molecules do notconsist of the exact sequence of the nucleotide sequences set forth inpublicly available databases, such as Expressed Sequence Tags (ESTs),Sequence Tagged Sites (STSs) and genomic fragments, deposited in publicdatabases such as the nr, dbest, dbsts, gss and htgs databases,including nucleotide sequences of the GI accession numbers set forthabove. Likewise, the invention CARD-10X, -11X and -12X polypeptides donot consist of the exact sequence of the amino acid sequences set forthin publicly available databases, or of the exact amino acid sequence ofa translated product of an EST set forth in the databases. Since one ofskill in the art will realize that the above-recited excluded sequencesmay be revised at a later date, the skilled artisan will recognize thatthe above-recited sequences are excluded as they stand on the prioritydate of this application.

The CARD domains of these CARD-10X, -11X and -12X are similar to eachother, sharing approximately 40-50% sequence identity, as well assimilarity by fold prediction criteria. The closest homologs of the CARDdomains of the disclosed CARD-containing polypeptides are found in humanB-cell CLL/lymphoma 10 protein, and in the equine herpesvirus 2hypothetical protein E10, which have about 25% sequence identity to thedisclosed CARD-domains.

Apart from their CARD domains, both CARD-10X and CARD-11X containconserved domains found in proteins that bind the cell cytoskeleton.Specifically, CARD-10X contains a domain of approximately 150 aminoacids found in many intermediate filament proteins (Pfam code 00038),designated the “filament” domain (SEQ ID NO:6). Intermediate filamentproteins are distinct but structurally related proteins that make upintermediate filaments, which are major components of the cytoskeleton.Many intermediate filament proteins are developmentally regulated andtissue specific, and undergo a variety of post-translationalmodifications, including phosphorylation.

CARD-11X contains a domain of approximately 300 amino acids similar to adomain found in members of the “Ezrin/radixin/moiesin” or “ERM” family(Pfam code 00769). The ERM domain of CARD-11X is most similar to thehelical linker region of ERM, but there is also some similarity to theactin-binding region of the ERM domain. ERM domain proteins are involvedin connections between cytoskeletal structures and the plasma membrane.ERM proteins mediate these connections by associating both with F-actinand with juxtamembrane proteins, including extracellular matrix and celladhesion receptors, in a regulated fashion.

CARD-11X also contains a domain designated the “post synaptic densitydisc-large zo-1” or “PDZ” domain (Pfam code 00595). PDZ domains areprotein interaction modules that mediate the binding of a class ofsubmembraneous proteins to ion channels and membrane receptors,including neurotransmitter receptors.

The structural and functional properties of intermediate filamentdomains, ERM domains and PDZ domains, and proteins containing suchdomains, can be found in the Pfam database. Pfam is a publicly availablelarge collection of multiple sequence alignments and hidden Markovmodels that covers many common protein domains. Version 5.5 of Pfam(September 2000) contains alignments and models for 2478 proteinfamilies, based on the Swissprot 38 and SP-TrEMBL 11 protein sequencedatabases.

CARD-10X and CARD-11X are the first CARD-containing polypeptidesdescribed to contain cytoskeleton binding domains. These proteins arelikely to provide a link between the cell cytoskeleton and the apoptosisregulatory machinery. In particular, CARD-10X and CARD-11X are proposedto play a role in the regulation and/or execution of anoikis, anapoptotic process that occurs when cells are deprived of attachments viaintegrins, causing disorganization of the cytoskeleton. Specifically, aCARD domain attached to the cytoskeleton may be involved in bringing acaspase to filaments in order to facilitate cytoskeleton cleavage.

This prediction is consistent with results indicating that at earlystages of apoptosis, caspases group near the cytoskeleton, andmicrovillar breakdown occurs. Additionally, a caspase-3-like proteasehas been show to play a role in the cleavage of the ERM protein moiesinduring platelet activation.

Accordingly, besides the biochemical processes such as apoptosis, NF-κBinduction, cytokine processing, cytokine receptor signaling, andcaspase-mediated proteolysis, in which other known CARD-containingpolypeptides are implicated, the CARD-containing polypeptides of theinvention are also predicted to be involved in cytoskeletal integrityand anoikis.

As is readily appreciated by one of skill in the art, different isoformsof a gene can be expressed as differently spliced gene products. Thus,it is contemplated herein that the CARD-containing polypeptides of theinvention can exist in a variety of isoforms. As referred to herein, an“isoform” of a CARD-containing polypeptide is a biologically activeCARD-containing polypeptide that contains at least two contiguous exonsequences from among the various exon sequences known to code for thereference polypeptide.

For example, CARD-11X (SEQ ID NO:8) is encoded by 26 exons, as evidencedby comparison of SEQ ID NO:7 with the genomic sequence set forth in theNR nucleotide database as GI accession number 9665194. CARD-12X (SEQ IDNO:16) is encoded by at least 2 exons, as evidenced by comparison of SEQID NO:15 with the genomic sequence set forth in the HTGS nucleotidedatabase as GI accession number 8224622. It is also contemplated hereinthat an isoform of a CARD-containing polypeptide can include additionalamino acids not encoded by the described exon sequences.

In one embodiment, the invention provides a substantially purifiedCARD-containing polypeptide, comprising substantially the same aminoacid sequence as the amino acid sequence of CARD-11X (SEQ ID NO:8) orCARD-12X (SEQ ID NO:16).

In another embodiment, the invention provides a substantially purifiedfunctional fragment of a CARD-containing polypeptide, comprisingsubstantially the same amino acid sequence as the amino acid sequence ofthe CARD domain of CARD-10X (SEQ ID NO:4), the filament domain ofCARD-10X (SEQ ID NO:6), the CARD domain of CARD-11X (SEQ ID NO:10) theERM (ezrin) domain of CARD-11X (SEQ ID NO:12), the PDZ domain ofCARD-11X (SEQ ID NO:14) and the CARD domain of CARD-12X (SEQ ID NO:16).

As employed herein, the term “substantially the same amino acidsequence” refers to amino acid sequences having at least about 70% or75% identity with respect to the reference amino acid sequence, andretaining comparable functional and biological activity characteristicof the polypeptide defined by the reference amino acid sequence.Preferably, polypeptides having “substantially the same amino acidsequence” will have at least about 80%, 82%, 84%, 86% or 88%, morepreferably 90%, 91%, 92%, 93% or 94% amino acid identity with respect tothe reference amino acid sequence; with greater than about 95%, 96%,97%, 98% or 99% amino acid sequence identity being especially preferred.

In accordance with the invention, specifically included within thedefinition of “substantially the same” amino acid sequence is thepredominant amino acid sequence of a particular inventionCARD-containing polypeptide disclosed herein. The predominant amino acidsequence refers to the most commonly expressed naturally occurring aminoacid sequence in a species population. A predominant polypeptide withmultiple isoforms will have the most commonly expressed amino acidsequence for each isoform. A predominant CARD-containing polypeptide ofthe invention refers to an amino acid sequence having sequence identityto an amino acid sequence disclosed herein that is greater than that ofany other naturally occurring protein of a particular species (e.g.,human).

Given the teachings herein of the nucleic acid or amino acid sequencescorresponding to the invention CARD-containing polypeptides, one ofskill in the art can readily confirm and, if necessary, revise thenucleic acid or amino acid sequences associated with the CARD-containingpolypeptides of the invention. For example, the sequences can beconfirmed by probing a cDNA library with a nucleic acid probecorresponding to a nucleic acid of the invention using PCR or otherknown methods. Further, an appropriate bacterial artificial chromosomecontaining the region of the genome encoding an inventionCARD-containing polypeptide can be commercially obtained and probedusing PCR, restriction mapping, sequencing, and other known methods.

A CARD-containing polypeptide or functional fragment thereof can haveconservative amino acid substitutions as compared with the referencepolypeptide amino acid sequence. Conservative substitutions of encodedamino acids include, for example, amino acids that belong within thefollowing groups: (1) non-polar amino acids (Gly, Ala, Val, Leu, andIle); (2) polar neutral amino acids (Cys, Met, Ser, Thr, Asn, and Gln);(3) polar acidic amino acids (Asp and Glu); (4) polar basic amino acids(Lys, Arg and His); and (5) aromatic amino acids (Phe, Trp, Tyr, andHis).

A CARD-containing polypeptide or functional fragment can also bechemically derivatized, provided that the polypeptide retains aCARD-containing polypeptide biological activity. For example, chemicalderivatization of an invention polypeptide can be alkylation, acylation,carbamoylation and iodination. Derivatized polypeptides also include,for example, those molecules in which free amino groups have beenderivatized to form amine hydrochlorides, p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups orformyl groups. Free carboxyl groups can be derivatized to form salts,methyl and ethyl esters or other types of esters or hydrazides. Freehydroxyl groups can be derivatized to form O-acyl or O-alkylderivatives. The imidazole nitrogen of histidine can be derivatized toform N-im-benzylhistidine.

A CARD-containing polypeptide or functional fragment can also besubstituted with one or more amino acid analogs of the twenty standardamino acids, for example, 4-hydroxyproline, 5-hydroxylysine,3-methylhistidine, homoserine, ornithine or carboxyglutamate, and caninclude amino acids that are not linked by peptide bonds.

A CARD-containing polypeptide or functional fragment can also containmimetic portions that orient functional groups that provide a functionof a CARD-containing polypeptide. Mimetics encompass chemicalscontaining chemical moieties that mimic the function of the polypeptide.For example, if a polypeptide contains two charged chemical moietieshaving functional activity, a mimetic places two charged chemicalmoieties in a spatial orientation and constrained structure so that thecharged chemical function is maintained in three-dimensional space.Exemplary mimetics are peptidomimetics, peptoids, or other peptide-likepolymers such as poly(β-amino acids), and also non-polymeric compoundsupon which functional groups that mimic a peptide are positioned.

Another embodiment of the invention provides a CARD-containingpolypeptide, or a functional fragment thereof, fused with a moiety toform a conjugate. As used herein, a “moiety” can be a physical, chemicalor biological entity which contributes functionality to aCARD-containing polypeptide or a functional fragment thereof.Functionalities contributed by a moiety include therapeutic or otherbiological activity, or the ability to facilitate identification orrecovery of a CARD-containing polypeptide. Therefore, a moiety willinclude molecules known in the art to be useful for detection of theconjugate by, for example, by fluorescence, magnetic imaging, detectionof radioactive emission. A moiety may also be useful for recovery of theconjugate, for example a His tag or other known tags used for proteinisolation and/or purification, or a physical substance such as a bead. Amoiety can be a therapeutic compound, for example, a cytotoxic drugwhich can be useful to effect a biological change in cells to which theconjugate localizes.

The term “functional”, when used herein as a modifier of inventionCARD-containing polypeptides, or fragments thereof, refers to apolypeptide that exhibits biological activities similar to at least aportion of a CARD-containing polypeptide of the invention. Biologicalactivities of a CARD-containing polypeptide of the invention include,for example, the ability to bind to a CARD-associated polypeptide (e.g.a caspase or pro-caspase), to another CARD-containing polypeptide, to acytoskeletal component, or to another protein, thereby alteringapoptosis, NF-κB induction, cytokine processing, cytokine receptorsignaling, caspase-mediated proteolysis, cytoskeletal integrity oranoikis.

The ability of a CARD-containing polypeptide to bind another polypeptidesuch as a CARD-associated polypeptide can be assayed, for example, usingmethods well known in the art, such as yeast two-hybrid assays,co-immunoprecipitation, fluorescence resonance energy transfer (FRET)assays, GST fusion co-purification, and the like.

In accordance with the invention, there are also provided functionalfragments of CARD-containing polypeptides which retain some, but notall, of the predominant naturally occurring CARD-containing polypeptideactivities. A “functional fragment” is any truncated form, eithercarboxy-terminal, amino-terminal, or both, of the predominant naturallyoccurring protein.

For example, a functional fragment of an invention polypeptide cancontain one or more of the following: a CARD domain, a filament domain,a ERM domain, and a PDZ domain. In a specific example, a functionalfragment of a CARD-containing polypeptide such as CARD-10X can contain aCARD domain, but lack a functional filament domain, or vice versa. Sucha fragment will retain certain CARD-10X biological activities (e.g.,CARD domain functionality), but not all such activities (e.g., lackfilament domain functionality).

In another example, a functional fragment of a CARD-11X polypeptide cancontain one, or any two, of the CARD domain, ERM domain and PDZ domains,but lack at least one functional domain. Such a fragment will retaincertain CARD-11X biological activities (e.g., CARD domain functionality,or association with cytoskeletal components, or both), but not all suchactivities.

In one embodiment, the activity of the functional fragment will be“dominant-negative.” A dominant-negative activity will allow thefragment to reduce or inactivate the activity of one or more isoforms ofa predominant naturally occurring CARD-containing polypeptide.

Another biological activity of a CARD-containing polypeptide orfunctional fragment thereof is the ability to act as an immunogen forthe production of polyclonal and monoclonal antibodies that bindspecifically to an invention CARD-containing polypeptide. Suchimmunologic activity may be assayed by any method known to those ofskill in the art. For example, a test-polypeptide encoded by aCARD-encoding cDNA can be used to produce antibodies, which are thenassayed for their ability to bind to an invention CARD-containingpolypeptide. If the antibody binds to the test-polypeptide and thereference polypeptide with substantially the same affinity, then thepolypeptide possesses the requisite immunologic biological activity.

Thus, the invention also provides a substantially purified functionalfragment of a CARD-containing polypeptide, comprising at least 10contiguous residues of CARD-10X (SEQ ID NO:2), CARD-11X (SEQ ID NO:8) orCARD-12X (SEQ ID NO:16), wherein the functional fragment is immunogenic.

The length of the functional fragments of the invention can range fromabout 10 amino acids up to the full-length sequence of an inventionCARD-containing polypeptide. In certain embodiments, such as forinvention immunogenic fragments, the amino acid lengths include, forexample, at least about 12 amino acids, such as at least about, or notmore than, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or 65 amino acids. Inother embodiments, such as for longer invention immunogenic fragmentsand fragments containing CARD, filament, ERM or PDZ domains, thefunctional fragments can contain at least about, or not more than, 70,75, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 or more aminoacids in length up to the full-length CARD-containing polypeptidesequence.

As used herein, the term “substantially purified” means a polypeptidethat is in a form that is relatively free from contaminating lipids,polypeptides, nucleic acids or other cellular material normallyassociated with a polypeptide in a cell. A substantially purifiedCARD-containing polypeptide can be obtained by a variety of methodswell-known in the art, e.g., recombinant expression systems describedherein, precipitation, gel filtration, ion-exchange, reverse-phase andaffinity chromatography, and the like. Other well-known methods aredescribed in Deutscher et al., “Guide to Protein Purification” Methodsin Enzymology Vol. 182, (Academic Press, (1990)). The methods andconditions for biochemical purification of a polypeptide of theinvention can be chosen by those skilled in the art, and purificationmonitored, for example, by an immunological assay, binding assay, or afunctional assay.

An example of a method for preparing the invention polypeptide(s) is toexpress nucleic acids encoding a CARD-containing polypeptide in asuitable host cell, such as a bacterial cell, a yeast cell, an amphibiancell such as an oocyte, or a mammalian cell, using methods well known inthe art, and recovering the expressed polypeptide, again usingwell-known purification methods. Invention polypeptides can be isolateddirectly from cells that have been transformed with expression vectorsas known in the art. Recombinantly expressed polypeptides of theinvention can also be expressed as fusion proteins with appropriateaffinity tags, such as glutathione S transferase (GST) or poly His, andaffinity purified. The invention polypeptide, biologically functionalfragments, and functional equivalents thereof can also be produced by invitro transcription/translation methods known in the art, such as usingreticulocyte lysates, as used for example, in the TNT system (Promega).The invention polypeptide, biologically functional fragments, andfunctional equivalents thereof can also be produced by chemicalsynthesis. For example, synthetic polypeptides can be produced usingApplied Biosystems, Inc. Model 430A or 431A automatic peptidesynthesizer (Foster City, Calif.) employing the chemistry provided bythe manufacturer.

Methods to identify additional invention polypeptides containing afunctional fragment of a CARD-containing polypeptide are well known inthe art. For example, genomic or cDNA libraries from any species ortissue are commercially available or can be readily prepared, and can beprobed according to methods known in the art. Full-lengthpolypeptide-encoding nucleic acids, such as full-length cDNAs can beobtained by a variety of methods well-known in the art, such as 5′ and3′ RACE.

In another embodiment of the invention, chimeric polypeptides areprovided comprising a CARD-containing polypeptide, or a functionalfragment thereof, fused with another polypeptide or functional fragmentthereof. Polypeptides with which the CARD-containing polypeptide orfunctional fragment thereof are fused can include, for example,glutathione-S-transferase, an antibody, or other proteins or functionalfragments thereof which facilitate recovery of the chimera. Furtherpolypeptides with which a CARD-containing polypeptide or functionalfragment thereof are fused will include, for example, luciferase, agreen fluorescent protein, an antibody, or other proteins or functionalfragments thereof which facilitate identification of the chimera. Stillfurther polypeptides with which a CARD-containing polypeptide orfunctional fragment thereof can advantageously be fused include, forexample, the LexA DNA binding domain, ricin, α-sarcin, an antibody orfragment thereof, or other polypeptides which have therapeuticproperties or other biological activity.

Further invention chimeric polypeptides contemplated herein are chimericpolypeptides wherein a functional fragment of a CARD-containingpolypeptide is fused with a catalytic domain or a protein interactiondomain from a heterologous polypeptide. One of skill in the art willappreciate that a large number of chimeric polypeptides are readilyavailable by combining domains of 2 or more CARD-containing polypeptidesof the invention. Further, chimeric polypeptides can contain afunctional fragment of a CARD-containing polypeptide of the inventionfused with a domain of a protein known in the art, such as CED-4,Apaf-1, caspase-1, and the like.

The invention also provides methods for administering CARD-containingpolypeptides to an individual to modulate an activity associated with aCARD-containing polypeptide, including induction of apoptosis oranoikis, tumor suppression, modulation of inflammation or cell adhesionand the like. A CARD-containing polypeptide can be administeredtherapeutically to an individual using expression vectors containingnucleic acids encoding CARD-containing polypeptides, as described below.In addition, CARD-containing polypeptides, or a functional portionthereof, can be directly administered to an individual. Methods ofadministering therapeutic polypeptides in the form of a pharmaceuticalcomposition are well known to those skilled in the art.

An exemplary method of delivering a CARD-containing polypeptide to anintracellular target is to fuse a CARD-containing polypeptide orfunctional fragment to an intracellular-targeting peptide that canpenetrate the cell membrane or otherwise deliver a polypeptide to theintracellular environment such as via internalization, thereby causingthe fused CARD-containing polypeptide to enter the cell. One example ofsuch an intracellular-targeting peptides is a fusion to the transductiondomain of HIV TAT, which allows transduction of up to 100% of cells(Schwarze et al., Science 285:1569-1572 (1999); Vocero-Akbani et al.,Nature Med. 5:29-33 (1999)).

Another example of such an intracellular-targeting peptide is theAntennapeida homeoprotein internalization domain (Holinger et al., J.Biol. Chem. 274:13298-13304 (1999)). Still anotherintracellular-targeting peptide is a peptide that is specific for a cellsurface receptor, which allows binding and internalization of a fusionpolypeptide via receptor-mediated endocytosis (Ellerby et al., NatureMed. 5:1032-1038 (1999)). Such intracellular-targeting peptides thatmediate specific receptor interactions can be advantageously used totarget a tumor (see Ellerby et al., supra, 1999). Alternatively, aCARD-containing polypeptide of the invention can be incorporated, ifdesired, into liposomes, microspheres or other polymer matrices(Gregoriadis, Liposome Technology, Vols. I to III, 2nd ed., CRC Press,Boca Raton Fla. (1993)).

In accordance with another embodiment of the invention, there areprovided isolated nucleic acid molecules encoding a CARD-containingpolypeptide or functional fragment thereof. The invention isolatednucleic acids encoding CARD-containing polypeptides are selected from:

(a) a nucleic acid molecule encoding a polypeptide comprising the aminoacid sequence of CARD-11X (SEQ ID NO:8) or CARD-12X (SEQ ID NO:16);

(b) a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO:7 or SEQ ID NO:15; and

(c) a nucleic acid molecule that hybridizes to the nucleic acid moleculeof (a) or (b) under moderately stringent hybridization conditions.

The invention also provides isolated nucleic acid molecules encodingfunctional fragments of a CARD-containing polypeptide selected from:

(a) a nucleic acid molecule encoding the CARD domain of CARD-10X (SEQ IDNO:4), the filament domain of CARD-10X (SEQ ID NO:6), the CARD domain ofCARD-11X (SEQ ID NO:10), the ERM (ezrin) domain of CARD-11X (SEQ IDNO:12), the PDZ domain of CARD-11X (SEQ. ID NO:14) and the CARD domainof CARD-12X (SEQ ID NO:16);

(b) a nucleic acid molecule comprising the nucleotide sequence of theCARD domain of CARD-10X (SEQ ID NO:3), the filament domain of CARD-10X(SEQ ID NO:5), the CARD domain of CARD-11X (SEQ ID NO:9), the ERM(ezrin) domain of CARD-11X (SEQ ID NO:11), the PDZ domain of CARD-11X(SEQ ID NO:13) and the CARD domain of CARD-12X (SEQ ID NO:15); and (c) anucleic acid molecule that hybridizes to the nucleic acid molecule of(a) or (b) under moderately stringent hybridization conditions.

Also provided are isolated nucleic acid molecules having substantiallythe same nucleotide sequence as the CARD-11X (SEQ ID NO:7) or CARD-12X(SEQ ID NO:15) coding sequence.

The nucleic acid molecules described herein are useful for producinginvention polypeptides, when such nucleic acids are incorporated into avariety of protein expression systems known to those of skill in theart. In addition, such nucleic acid molecules or fragments thereof canbe labeled with a readily detectable substituent and used ashybridization probes for assaying for the presence and/or amount of aninvention CARD-encoding gene or mRNA transcript in a given sample. Thenucleic acid molecules described herein, and fragments thereof, are alsouseful as primers and/or templates in a PCR reaction for amplifyinggenes encoding invention polypeptides described herein.

The term “nucleic acid molecule” or “polynucleotide” encompassesribonucleic acid (RNA) or deoxyribonucleic acid (DNA), probes,oligonucleotides, and primers, and can be single stranded or doublestranded. DNA can be either complementary DNA (cDNA) or genomic DNA,e.g. a CARD-encoding gene, and can represent the sense strand, theanti-sense strand, or both. Examples of nucleic acids are RNA, cDNA, andisolated genomic DNA encoding a CARD-containing polypeptide.

One means of isolating a CARD-encoding nucleic acid polypeptide is toprobe a mammalian genomic or cDNA library with a natural or artificiallydesigned DNA probe using methods well known in the art. DNA probesderived from the CARD-encoding gene are particularly useful for thispurpose. DNA and cDNA molecules that encode CARD-containing polypeptidescan be used to obtain complementary genomic DNA, cDNA or RNA frommammalian (e.g., human, mouse, rat, rabbit, pig, and the like), or otheranimal sources, or to isolate related cDNA or genomic clones byscreening cDNA or genomic libraries, using methods described in moredetail below.

In general, a genomic sequence of the invention includes regulatoryregions such as promoters, enhancers, and introns that are outside ofthe exons encoding a CARD-containing polypeptide, but does not includeproximal genes that do not encode a CARD-containing polypeptide.

Use of the term “isolated” as a modifier of nucleic acids orpolypeptides means that the molecules so designated have been producedin such form by the hand of man, and thus are separated from theirnative in vivo cellular environment.

Invention nucleic acids encoding CARD-containing polypeptides andinvention CARD-containing polypeptides can be obtained from any speciesof organism, such as prokaryotes, eukaryotes, plants, fungi,vertebrates, invertebrates, and the like. A preferred source ofinvention nucleic acids are mammalian species, e.g., human, rat, mouse,rabbit, monkey, baboon, bovine, porcine, ovine, canine, feline, and thelike, with human particularly preferred.

As employed herein, the term “substantially the same nucleotidesequence” refers to a nucleic acid molecule having sufficient identityto the reference polynucleotide, such that it will hybridize to thereference polynucleotide under moderately or highly stringenthybridization conditions. In one embodiment, a nucleic acid moleculehaving substantially the same nucleotide sequence as the referencenucleotide sequence encodes substantially the same amino acid sequenceas that set forth in any of SEQ ID NOS:2, 8 or 16, or its functionalfragments. In another embodiment, DNA having “substantially the samenucleotide sequence” as the reference nucleotide sequence has at least60%, such as 65%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86% or 88%,more preferably at least 90%, 91%, 92%, 93% or 94%, yet more preferablyat least 95%, 96%, 97%, 98% or 99% identity to the reference nucleotidesequence.

In accordance with the invention, specifically included within thedefinition of “substantially the same” nucleotide sequence is thepredominant nucleotide sequence of a particular inventionCARD-containing polypeptide described herein. The predominant nucleotidesequence refers to the most commonly present naturally occurringnucleotide sequence in a species population. A predominant CARD-encodingnucleic acid of the invention refers to a nucleotide sequence havingsequence identity to a nucleotide sequence disclosed herein that isgreater than that of any other naturally occurring nucleotide sequenceof a particular species (e.g., human).

A nucleotide sequence that is substantially the same as a referencenucleotide sequence can include, for example, one or several nucleotideadditions, deletions, or substitutions with respect to the referencesequence. Exemplary substitutions to a reference sequence aresubstitutions that do not change the encoded amino acid sequence due tothe degeneracy of the genetic code, or that result in a nucleotidesequence that encodes an amino acid sequence that is “substantially thesame” as a reference polypeptide, as described above. Such additions,deletions and substitutions can correspond to variations that are madedeliberately, or which occur as mutations during nucleic acidreplication.

A nucleotide sequence that is substantially the same as a referenceCARD-encoding nucleotide sequence can be a sequence that corresponds tohomologs of other species, including other mammalian species. Thecorresponding nucleotide sequences of non-human species can bedetermined by methods known in the art, such as by PCR or by screeninggenomic, cDNA or expression libraries. A nucleotide sequence that issubstantially the same as a reference sequence can also correspond tosplice variant forms of the CARD-encoding nucleotide sequence.

A nucleic acid molecule of the invention can include one or morenon-native nucleotides, having, for example, modifications to the base,the sugar, or the phosphate portion, or having a modified phosphodiesterlinkage. Such modifications can be advantageous in increasing thestability of the nucleic acid molecule.

Furthermore, a nucleic acid molecule of the invention can include, forexample, a detectable moiety, such as a radiolabel, a fluorochrome, aferromagnetic substance, a luminescent tag or a detectable binding agentsuch as biotin. Such moieties can be advantageous in applications wheredetection of a CARD-encoding nucleic acid molecule is desired.

As used herein, the term “hybridization” refers to the binding ofcomplementary strands of nucleic acid (i.e., sense:antisense strands orprobe:target-DNA) to each other through hydrogen bonds, similar to thebonds that naturally occur in chromosomal DNA. Stringency levels used tohybridize a given probe with target-DNA can be readily varied by thoseof skill in the art.

The phrase “stringent hybridization” is used herein to refer toconditions under which polynucleic acid hybrids are stable. As known tothose of skill in the art, the stability of hybrids is reflected in themelting temperature (Tm) of the hybrids. In general, the stability of ahybrid is a function of sodium ion concentration and temperature.Typically, the hybridization reaction is performed under conditions oflower stringency, followed by washes of varying, but higher, stringency.Reference to hybridization stringency relates to such washingconditions.

As used herein, the phrase “moderately stringent hybridization” refersto conditions that permit target-nucleic acid to bind a complementarynucleic acid. The hybridized nucleic acids will generally have at leastabout 60% identity, such as at least about 75% identity, more preferablyat least about 90% identity. Moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C.

The phrase “high stringency hybridization” refers to conditions thatpermit hybridization of only those nucleic acid sequences that formstable hybrids in 0.018M NaCl at 65° C. Thus, if a hybrid is not stablein 0.018M NaCl at 65° C., it will not be stable under high stringencyconditions, as contemplated herein. High stringency conditions can beprovided, for example, by hybridization in 50% formamide, 5×Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1X SSPE,and 0.1% SDS at 65° C.

Other suitable moderate stringency and high stringency hybridizationbuffers and conditions are well known to those of skill in the art andare described, for example, in Sambrook et al., Molecular Cloning—ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., (1989), and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., (Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (most recentSupplement).

Identity of any two nucleic acid or amino acid sequences can bedetermined by those skilled in the art based, for example, on a BLAST2.0 computer alignment, using default parameters. BLAST 2.0 computersearching and sequence alignments are known in the art (e.g. Tatusova etal., FEMS Microbiol Lett. 174:247-250 (1999) and Altschul et al.,Nucleic Acids Res., 25:3389-3402 (1997)), and are publicly available.

One means of isolating a nucleic acid encoding a CARD-containingpolypeptide is to probe a cDNA library or genomic library with a naturalor artificially designed nucleic acid probe using methods well known inthe art. Nucleic acid probes derived from a CARD-encoding gene areparticularly useful for this purpose. DNA and cDNA molecules that encodeCARD-containing polypeptides can be used to obtain complementary genomicDNA, cDNA or RNA from mammals, for example, human, mouse, rat, rabbit,pig, and the like, or other animal sources, or to isolate related cDNAor genomic clones by the screening of cDNA or genomic libraries, bymethods well known in the art.

The invention further provides oligonucleotides that function as singlestranded nucleic acid primers for amplification of a CARD-encodingnucleic acid, or as probes for identification of a CARD-encoding nucleicacid.

The invention oligonucleotides comprise at least 15 contiguousnucleotides of SEQ ID NOS:1, 7 or 15, or its complement. The inventionoligonucleotides can include at least, or not more than, 16, 17, 18, 19,20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250,275, 300, 325, 350 or more contiguous nucleotides from the referencenucleotide sequence or its complement.

The oligonucleotides of the invention are able to hybridize to thereference nucleic acid molecules of the invention under moderatelystringent, or highly stringent, hybridization conditions and thus can beadvantageously used, for example, as probes to detect CARD-encoding DNAor RNA in a sample, and to detect splice variants thereof; as sequencingor PCR primers; as antisense reagents to block transcription ofCARD-encoding RNA in cells; or in other applications known to thoseskilled in the art in which hybridization to a CARD-encoding nucleicacid molecule is desirable.

In accordance with another embodiment of the invention, a method isprovided for identifying nucleic acids encoding a CARD-containingpolypeptide. The method includes the steps of contacting a samplecontaining nucleic acids with an invention oligonucleotide, wherein thecontacting is effected under high stringency hybridization conditions,and identifying a nucleic acid that hybridizes thereto.

The invention additionally provides a method of detecting aCARD-encoding nucleic acid molecule in a sample by contacting the samplewith two or more invention oligonucleotides, amplifying a nucleic acidmolecule, and detecting the amplification. The amplification can beperformed, for example, using PCR.

The isolated nucleic acid molecules an oligonucleotides of the inventioncan be used in a variety of diagnostic and therapeutic applications. Forexample, the isolated nucleic acid molecules of the invention can beused as probes, as described above; as templates for the recombinantexpression of CARD-containing polypeptides; or in screening assays suchas two-hybrid assays to identify cellular molecules that bindCARD-containing polypeptides.

The isolated nucleic acid molecules of the invention can be prepared bymethods known in the art. The method chosen will depend on factors suchas the type and size of nucleic acid molecule one intends to isolate;whether or not it encodes a biologically active polypeptide (e.g. havinga CARD-containing polypeptide biological or immunogenic activity); andthe source of the nucleic acid molecule.

An exemplary method for preparing an isolated nucleic acid moleculeinvolves amplification of the nucleic acid molecule using inventionoligonucleotide primers and the polymerase chain reaction (PCR) and,optionally, purification of the resulting product by gelelectrophoresis. Using PCR, a CARD-encoding nucleic acid molecule havingany desired boundaries can be amplified exponentially starting from onlya few DNA or RNA molecules, such as from a single cell. Desiredmodifications to the nucleic acid sequence can also be introduced bychoosing an appropriate oligonucleotide primer with one or moreadditions, deletions or substitutions. PCR methods, including methods ofisolating homologs of a given nucleic acid molecule in another speciesusing degenerate primers, are well known in the art.

Alternatively, an isolated CARD-encoding nucleic acid molecule can beprepared by screening a library, such as a genomic library, cDNA libraryor expression library, with a detectable CARD-encoding nucleic acidmolecule or anti-CARD antibody. Human libraries, and libraries from alarge variety of mammalian species, are commercially available or can beproduced from species or cells of interest. The library clonesidentified as containing CARD-encoding nucleic acid molecules can beisolated, subcloned or sequenced by routine methods.

Furthermore, an isolated CARD-encoding nucleic acid molecule oroligonucleotide can be prepared by direct synthetic methods. Forexample, a single stranded nucleic acid molecule can be chemicallysynthesized in one piece, or in several pieces, by automated synthesismethods known in the art. The complementary strand can likewise besynthesized in one or more pieces, and a double-stranded molecule madeby annealing the complementary strands. Direct synthesis is particularlyadvantageous for producing relatively short molecules, such as probesand primers, and also for producing nucleic acid molecules containingmodified nucleotides or linkages.

The invention also provides methods for detecting a CARD-encodingnucleic acid in a sample. The methods of detecting a CARD-encodingnucleic acid in a sample can be either qualitative or quantitative, asdesired. For example, the presence, abundance, integrity or structure ofa CARD-encoding nucleic acid can be determined, as desired, depending onthe assay format and the probe used for hybridization or primer pairchosen for application.

Useful assays for detecting a CARD-containing nucleic acid based onspecific hybridization with an isolated invention oligonucleotide arewell known in the art and include, for example, in situ hybridization,which can be used to detect altered chromosomal location of the nucleicacid molecule, altered gene copy number, and RNA abundance, depending onthe assay format used. Other hybridization assays include, for example,Northern blots and RNase protection assays, which can be used todetermine the abundance and integrity of different RNA splice variants,and Southern blots, which can be used to determine the copy number andintegrity of DNA. A hybridization probe can be labeled with any suitabledetectable moiety, such as a radioisotope, fluorochrome,chemiluminescent marker, biotin, or other detectable moiety known in theart that is detectable by analytical methods.

Useful assays for detecting a CARD-encoding nucleic acid in a samplebased on amplifying a CARD-encoding nucleic acid with two or moreinvention oligonucleotides are also well known in the art, and include,for example, qualitative or quantitative polymerase chain reaction(PCR); reverse-transcription PCR (RT-PCR); single strand conformationalpolymorphism (SSCP) analysis, which can readily identify a single pointmutation in DNA based on differences in the secondary structure ofsingle-strand DNA that produce an altered electrophoretic mobility uponnon-denaturing gel electrophoresis; and coupled PCR, transcription andtranslation assays, such as a protein truncation test, in which amutation in DNA is determined by an altered protein product on anelectrophoresis gel. Additionally, the amplified CARD-encoding nucleicacid can be sequenced to detect mutations and mutational hot-spots, andspecific assays for large-scale screening of samples to identify suchmutations can be developed.

Also provided are antisense-nucleic acids having a sequence capable ofbinding specifically with full-length or any portion of an mRNA thatencodes CARD-containing polypeptides so as to prevent translation of themRNA. The antisense-nucleic acid can have a sequence capable of bindingspecifically with any portion of the sequence of the cDNA encodingCARD-containing polypeptides. As used herein, the phrase “bindingspecifically” encompasses the ability of a nucleic acid sequence torecognize a complementary nucleic acid sequence and to formdouble-helical segments therewith via the formation of hydrogen bondsbetween the complementary base pairs. An example of an antisense-nucleicacid is an antisense-nucleic acid comprising chemical analogs ofnucleotides.

The present invention provides means to alter levels of expression ofCARD-containing polypeptides by recombinantly expressing CARD-containinganti-sense nucleic acids or employing synthetic anti-sense nucleic acidcompositions (hereinafter SANC) that inhibit translation of mRNAencoding these polypeptides. Synthetic oligonucleotides, or otherantisense-nucleic acid chemical structures designed to recognize andselectively bind to mRNA are constructed to be complementary tofull-length or portions of a CARD-encoding strand, including nucleotidesequences substantially the same as SEQ ID NOS:1, 7 or 15.

The SANC is designed to be stable in the blood stream for administrationto a subject by injection, or in laboratory cell culture conditions. TheSANC is designed to be capable of passing through the cell membrane inorder to enter the cytoplasm of the cell by virtue of physical andchemical properties of the SANC, which render it capable of passingthrough cell membranes, for example, by designing small, hydrophobicSANC chemical structures, or by virtue of specific transport systems inthe cell which recognize and transport the SANC into the cell. Inaddition, the SANC can be designed for administration only to certainselected cell populations by targeting the SANC to be recognized byspecific cellular uptake mechanisms which bind and take up the SANC onlywithin select cell populations. In a particular embodiment the SANC isan antisense oligonucleotide.

For example, the SANC may be designed to bind to a receptor found onlyin a certain cell type, as discussed above. The SANC is also designed torecognize and selectively bind to target mRNA sequence, which cancorrespond to a sequence contained within the sequences set forth as SEQID NOS:1, 7 or 15.

The SANC is designed to inactivate target mRNA sequence by eitherbinding thereto and inducing degradation of the mRNA by, for example,RNase I digestion, or inhibiting translation of mRNA target sequence byinterfering with the binding of translation-regulating factors orribosomes, or inclusion of other chemical structures, such as ribozymesequences or reactive chemical groups which either degrade or chemicallymodify the target mRNA. SANCs have been shown to be capable of suchproperties when directed against mRNA targets (see Cohen et al., TIPS,10:435 (1989) and Weintraub, Sci. American, January (1990), pp. 40).

The invention further provides a method of altering the level of abiochemical process modulated by a CARD-containing polypeptide byintroducing an antisense nucleotide sequence into the cell, wherein theantisense nucleotide sequence specifically hybridizes to a CARD-encodingnucleic acid molecule, wherein the hybridization reduces or inhibits theexpression of the CARD-containing polypeptide in the cell. The use ofanti-sense nucleic acids, including recombinant anti-sense nucleic acidsor SANCs, can be advantageously used to inhibit cell death.

Compositions comprising an amount of the antisense-nucleic acid of theinvention, effective to reduce expression of CARD-containingpolypeptides by entering a cell and binding specifically toCARD-encoding mRNA so as to prevent translation and an acceptablehydrophobic carrier capable of passing through a cell membrane are alsoprovided herein. Suitable hydrophobic carriers are described, forexample, in U.S. Pat. Nos. 5,334,761; 4,889,953; 4,897,355, and thelike. The acceptable hydrophobic carrier capable of passing through cellmembranes may also comprise a structure which binds to a receptorspecific for a selected cell type and is thereby taken up by cells ofthe selected cell type. For example, the structure can be part of aprotein known to bind to a cell-type specific receptor such as a tumor.

Antisense-nucleic acid compositions are useful to inhibit translation ofmRNA encoding invention polypeptides. Synthetic oligonucleotides, orother antisense chemical structures are designed to bind toCARD-encoding mRNA and inhibit translation of mRNA and are useful ascompositions to inhibit expression of CARD-encoding genes orCARD-associated polypeptide genes in a tissue sample or in a subject.

The above-described nucleotide sequences can be incorporated intovectors for further manipulation. As used herein, vector refers to arecombinant DNA or RNA plasmid or virus containing discrete elementsthat are used to introduce heterologous DNA into cells for eitherexpression or replication thereof.

The invention also provides vectors containing the CARD-encoding nucleicacids of the invention. Suitable expression vectors are well-known inthe art and include vectors capable of expressing nucleic acidoperatively linked to a regulatory sequence or element such as apromoter region or enhancer region that is capable of regulatingexpression of such nucleic acid. Appropriate expression vectors includethose that are replicable in eukaryotic cells and/or prokaryotic cellsand those that remain episomal or those which integrate into the hostcell genome.

Promoters or enhancers, depending upon the nature of the regulation, canbe constitutive or regulated. The regulatory sequences or regulatoryelements are operatively linked to a nucleic acid of the invention suchthat the physical and functional relationship between the nucleic acidand the regulatory sequence allows transcription of the nucleic acid.

Suitable vectors for expression in prokaryotic or eukaryotic cells arewell known to those skilled in the art (see, for example, Ausubel etal., supra, 2000). Vectors useful for expression in eukaryotic cells caninclude, for example, regulatory elements including the SV40 earlypromoter, the cytomegalovirus (CMV) promoter, the mouse mammary tumorvirus (MMTV) steroid-inducible promoter, Moloney murine leukemia virus(MMLV) promoter, and the like. The vectors of the invention are usefulfor subcloning and amplifying a CARD-encoding nucleic acid molecule andfor recombinantly expressing a CARD-containing polypeptide. A vector ofthe invention can include, for example, viral vectors such as abacteriophage, a baculovirus or a retrovirus; cosmids or plasmids; and,particularly for cloning large nucleic acid molecules, bacterialartificial chromosome vectors (BACs) and yeast artificial chromosomevectors (YACs). Such vectors are commercially available, and their usesare well known in the art. One skilled in the art will know or canreadily determine an appropriate promoter for expression in a particularhost cell.

The invention additionally provides recombinant cells containingCARD-encoding nucleic acids of the invention. The recombinant cells aregenerated by introducing into a host cell a vector containing aCARD-encoding nucleic acid molecule. The recombinant cells aretransducted, transfected or otherwise genetically modified. Exemplaryhost cells that can be used to express recombinant CARD moleculesinclude mammalian primary cells; established mammalian cell lines, suchas COS, CHO, HeLa, NIH3T3, HEK 293 and PC12 cells; amphibian cells, suchas Xenopus embryos and oocytes and other vertebrate cells. Exemplaryhost cells also include insect cells such as Drosophila, yeast cellssuch as Saccharomyces cerevisiae, Saccharomyces pombe, or Pichiapastoris, and prokaryotic cells such as Escherichia coli. Additionalhost cells can be obtained, for example, from ATCC (Manassas, Va.).

The invention also provides a method for expression of a CARD-containingpolypeptide by culturing cells containing a CARD-encoding nucleic acidunder conditions suitable for expression of a CARD-containingpolypeptide. Suitable culturing conditions for expression of an encodednucleic acid molecule are well known in the art, and described, forexample, in Ausubel et al., supra, 2000.

CARD-encoding nucleic acids can also be delivered into mammalian cells,either in vivo or in vitro, to modulate an activity associated with aCARD-containing polypeptide, including induction of apoptosis oranoikis, tumor suppression, modulation of inflammation or cell adhesionand the like. Suitable vectors for delivering a CARD-encoding nucleicacid molecule of the invention to a mammalian cell include viral vectorssuch as retroviral vectors, adenovirus, adeno-associated virus,lentivirus, herpesvirus, as well as non-viral vectors such as plasmidvectors. Such vectors are useful for providing therapeutic amounts of aCARD-containing polypeptide (see, for example, U.S. Pat. No. 5,399,346,issued Mar. 21, 1995). Delivery of CARD nucleic acids therapeuticallycan be particularly useful when targeted to a tumor cell, therebyinducing apoptosis in tumor cells. In addition, where it is desirable tolimit or reduce the in vivo expression of a CARD-containing polypeptide,the introduction of a vector expressing the antisense strand of theinvention nucleic acid molecule is contemplated.

Viral based systems provide the advantage of being able to introducerelatively high levels of the heterologous nucleic acid into a varietyof cells. Suitable viral vectors for introducing an inventionCARD-encoding nucleic acid into mammalian cells are well known in theart. These viral vectors include, for example, Herpes simplex virusvectors (Geller et al., Science, 241:1667-1669 (1988)); vaccinia virusvectors (Piccini et al., Meth. Enzymology, 153:545-563 (1987));cytomegalovirus vectors (Mocarski et al., in Viral Vectors, Y. Gluzmanand S. H. Hughes, Eds., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1988, pp. 78-84)); Moloney murine leukemia virus vectors(Danos et al., Proc. Natl. Acad. Sci. USA, 85:6460-6464 (1988); Blaeseet al., Science, 270:475-479 (1995); Onodera et al., J. Virol.,72:1769-1774 (1998)); adenovirus vectors (Berkner, Biotechniques,6:616-626 (1988); Cotten et al., Proc. Natl. Acad. Sci. USA,89:6094-6098 (1992); Graham et al., Meth. Mol. Biol., 7:109-127 (1991);Li et al., Human Gene Therapy, 4:403-409 (1993); Zabner et al., NatureGenetics, 6:75-83 (1994)); adeno-associated virus vectors (Goldman etal., Human Gene Therapy, 10:2261-2268 (1997); Greelish et al., NatureMed., 5:439-443 (1999); Wang et. al., Proc. Natl. Acad. Sci. USA,96:3906-3910 (1999); Snyder et al., Nature Med., 5:64-70 (1999); Herzoget al., Nature Med., 5:56-63 (1999)); retrovirus vectors (Donahue etal., Nature Med., 4:181-186 (1998); Shackleford et al., Proc. Natl.Acad. Sci. USA, 85:9655-9659 (1988); U.S. Pat. Nos. 4,405,712, 4,650,764and 5,252,479, and WIPO publications WO 92/07573, WO 90/06997, WO89/05345, WO 92/05266 and WO 92/14829; and lentivirus vectors (Kafri etal., Nature Genetics, 17:314-317 (1997)).

For example, in one embodiment of the present invention,adenovirus-transferrin/polylysine-DNA (TfAdpl-DNA) vector complexes(Wagner et al., Proc. Natl. Acad. Sci. USA, 89:6099-6103 (1992); Curielet al., Hum. Gene Ther., 3:147-154 (1992); Gao et al., Hum. Gene Ther.,4:14-24 (1993)) are employed to transduce mammalian cells withheterologous CARD-encoding nucleic acid. Any of the plasmid expressionvectors described herein may be employed in a TfAdpl-DNA complex.

Vectors useful for therapeutic administration of a CARD-encoding nucleicacid can contain a regulatory element that provides tissue specific orinducible expression of an operatively linked nucleic acid. One skilledin the art can readily determine an appropriate tissue-specific promoteror enhancer that allows expression of a CARD polypeptide or nucleic acidin a desired tissue. Any of a variety of inducible promoters orenhancers can also be included in the vector for regulatable expressionof a CARD polypeptide or nucleic acid. Such inducible systems, include,for example, tetracycline inducible system (Gossen & Bizard, Proc. Natl.Acad. Sci. USA, 89:5547-5551 (1992); Gossen et al., Science,268:1766-1769 (1995); Clontech, Palo Alto, Calif.); metallothioneinpromoter induced by heavy metals; insect steroid hormone responsive toecdysone or related steroids such as muristerone (No et al., Proc. Natl.Acad. Sci. USA, 93:3346-3351 (1996); Yao et al., Nature, 366:476-479(1993); Invitrogen, Carlsbad, Calif.); mouse mammary tumor virus (MMTV)induced by steroids such as glucocorticoid and estrogen (Lee et al.,Nature, 294:228-232 (1981); and heat shock promoters inducible bytemperature changes.

An inducible system particularly useful for therapeutic administrationutilizes an inducible promoter that can be regulated to deliver a levelof therapeutic product in response to a given level of drug administeredto an individual and to have little or no expression of the therapeuticproduct in the absence of the drug. One such system utilizes a Gal4fusion that is inducible by an antiprogestin such as mifepristone in amodified adenovirus vector (Burien et al., Proc. Natl. Acad. Sci. USA,96:355-360 (1999). Another such inducible system utilizes the drugrapamycin to induce reconstitution of a transcriptional activatorcontaining rapamycin binding domains of FKBP12 and FRAP in anadeno-associated virus vector (Ye et al., Science, 283:88-91 (1999)). Itis understood that any combination of an inducible system can becombined in any suitable vector, including those disclosed herein. Sucha regulatable inducible system is advantageous because the level ofexpression of the therapeutic product can be controlled by the amount ofdrug administered to the individual or, if desired, expression of thetherapeutic product can be terminated by stopping administration of thedrug.

The specificity of viral vectors for particular cell types can beutilized to target predetermined cell types. Thus, the selection of aviral vector will depend, in part, on the cell type to be targeted. Forexample, if a neurodegenerative disease is to be treated by increasingthe level of a CARD-containing polypeptide in neuronal cells affected bythe disease, then a viral vector that targets neuronal cells can beused. A vector derived from a herpes simplex virus is an example of aviral vector that targets neuronal cells (Battleman et al., J. Neurosci.13:941-951 (1993), which is incorporated herein by reference).Similarly, if a disease or pathological condition of the hematopoieticsystem is to be treated, then a viral vector that is specific for aparticular blood cell or its precursor cell can be used. A vector basedon a human immunodeficiency virus is an example of such a viral vector(Carroll et al., J. Cell. Biochem. 17E:241 (1993), which is incorporatedherein by reference). In addition, a viral vector or other vector can beconstructed to express a CARD-encoding nucleic acid in a tissue specificmanner by incorporating a tissue-specific promoter or enhancer into thevector (Dai et al., Proc. Natl. Acad. Sci. USA 89:10892-10895 (1992),which is incorporated herein by reference).

For gene therapy, a vector containing a CARD-encoding nucleic acid or anantisense nucleotide sequence can be administered to a subject byvarious methods. For example, if viral vectors are used, administrationcan take advantage of the target specificity of the vectors. In suchcases, there in no need to administer the vector locally at the diseasedsite. However, local administration can be a particularly effectivemethod of administering a CARD-encoding nucleic acid. In addition,administration can be via intravenous or subcutaneous injection into thesubject. Following injection, the viral vectors will circulate untilthey recognize host cells with the appropriate target specificity forinfection. Injection of viral vectors into the spinal fluid also can bean effective mode of administration, for example, in treating aneurodegenerative disease.

Receptor-mediated DNA delivery approaches also can be used to deliver aCARD-encoding nucleic acid molecule into cells in a tissue-specificmanner using a tissue-specific ligand or an antibody that isnon-covalently complexed with the nucleic acid molecule via a bridgingmolecule (Curiel et al., Hum. Gene Ther. 3:147-154 (1992); Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987), each of which is incorporated hereinby reference). Direct injection of a naked or a nucleic acid moleculeencapsulated, for example, in cationic liposomes also can be used forstable gene transfer into non-dividing or dividing cells in vivo (Ulmeret al., Science 259:1745-1748 (1993), which is incorporated herein byreference). In addition, a CARD-encoding nucleic acid molecule can betransferred into a variety of tissues using the particle bombardmentmethod (Williams et al., Proc. Natl. Acad. Sci. USA 88:2726-2730 (1991),which is incorporated herein by reference). Such nucleic acid moleculescan be linked to the appropriate nucleotide sequences required fortranscription and translation.

A particularly useful mode of administration of a CARD-encoding nucleicacid is by direct inoculation locally at the site of the disease orpathological condition. Local administration can be advantageous becausethere is no dilution effect and, therefore, the likelihood that amajority of the targeted cells will be contacted with the nucleic acidmolecule is increased. Thus, local inoculation can alleviate thetargeting requirement necessary with other forms of administration and,if desired, a vector that infects all cell types in the inoculated areacan be used. If expression is desired in only a specific subset of cellswithin the inoculated area, then a promoter, an enhancer or otherexpression element specific for the desired subset of cells can belinked to the nucleic acid molecule. Vectors containing such nucleicacid molecules and regulatory elements can be viral vectors, viralgenomes, plasmids, phagemids and the like. Transfection vehicles such asliposomes also can be used to introduce a non-viral vector intorecipient cells. Such vehicles are well known in the art.

The invention additionally provides an isolated anti-CARD antibodyhaving specific reactivity with a invention CARD-containing polypeptide.The anti-CARD antibody can be a monoclonal antibody or a polyclonalantibody. The invention further provides cell lines producing monoclonalantibodies having specific reactivity with an invention CARD-containingprotein.

The invention thus provides antibodies that specifically bind aCARD-containing polypeptide. CARD-specific antibodies be used, forexample, for the immunoaffinity or affinity chromatography purificationof an invention CARD-containing polypeptide, as well as for diagnosticand in vivo imaging procedures.

As used herein, the term “antibody” is used in its broadest sense toinclude polyclonal and monoclonal antibodies, as well as antigen bindingfragments of such antibodies. With regard to an anti-CARD antibody ofthe invention, the term “antigen” means a native or synthesizedCARD-containing polypeptide or fragment thereof. An anti-CARD antibody,or antigen binding fragment of such an antibody, is characterized byhaving specific binding activity for a CARD polypeptide or a peptideportion thereof of at least about 1×10⁵ M⁻¹. Thus, Fab, F(ab′)₂, Fd andFv fragments of an anti-CARD antibody, which retain specific bindingactivity for a CARD-containing polypeptide, are included within thedefinition of an antibody. Specific binding activity of aCARD-containing polypeptide can be readily determined by one skilled inthe art, for example, by comparing the binding activity of an anti-CARDantibody to a CARD-containing polypeptide versus a reference polypeptidethat is not a CARD-containing polypeptide. Methods of preparingpolyclonal or monoclonal antibodies are well known to those skilled inthe art (see, for example, Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press (1988)).

In addition, the term “antibody” as used herein includes naturallyoccurring antibodies as well as non-naturally occurring antibodies,including, for example, single chain antibodies, chimeric, bifunctionaland humanized antibodies, as well as antigen-binding fragments thereof.Such non-naturally occurring antibodies can be constructed using solidphase peptide synthesis, can be produced recombinantly or can beobtained, for example, by screening combinatorial libraries consistingof variable heavy chains and variable light chains as described by Huseet al., Science 246:1275-1281 (1989)). These and other methods ofmaking, for example, chimeric, humanized, CDR-grafted, single chain, andbifunctional antibodies are well known to those skilled in the art(Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al.,Nature 341:544-546 (1989); Harlow and Lane, supra, 1988); Hilyard etal., Protein Engineering: A practical approach (IRL Press 1992);Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995)).

Anti-CARD antibodies can be raised using a CARD immunogen such as anisolated CARD-containing polypeptide having substantially the same aminoacid sequence as SEQ ID NOS:2, 8 or 15, or an immunogenic fragmentthereof, which can be prepared from natural sources or producedrecombinantly, or a peptide portion of the CARD-containing polypeptide.A non-immunogenic or weakly immunogenic CARD-containing polypeptide orportion thereof can be made immunogenic by coupling the hapten to acarrier molecule such as bovine serum albumin (BSA) or keyhole limpethemocyanin (KLH). Various other carrier molecules and methods forcoupling a hapten to a carrier molecule are well known in the art (see,for example, Harlow and Lane, supra, 1988). An immunogenicCARD-containing polypeptide fragment can also be generated by expressingthe peptide as a fusion protein, for example, to glutathione Stransferase (GST), polyHis or the like. Methods for expressing peptidefusions are well known to those skilled in the art (Ausubel et al.,supra, (2000)).

The invention further provides a method for detecting the presence of ahuman CARD-containing polypeptide in a sample by contacting a samplewith a CARD-specific antibody, and detecting the presence of specificbinding of the antibody to the sample, thereby detecting the presence ofa human CARD-containing polypeptide in the sample. CARD-specificantibodies can be used in diagnostic methods and systems to detect thelevel of CARD-containing polypeptide present in a sample. With respectto the detection of such polypeptides, the antibodies can be used for invitro diagnostic or in vivo imaging methods.

As used herein, the term “sample” is intended to mean any biologicalfluid, cell, tissue, organ or portion thereof, that includes orpotentially includes CARD nucleic acids or polypeptides. The termincludes samples present in an individual as well as samples obtained orderived from the individual. For example, a sample can be a histologicsection of a specimen obtained by biopsy, or cells that are placed in oradapted to tissue culture. A sample further can be a subcellularfraction or extract, or a crude or substantially pure nucleic acid orpolypeptide preparation.

Immunological procedures useful for in vitro detection of targetCARD-containing polypeptides in a sample include immunoassays thatemploy a detectable antibody. Such immunoassays include, for example,immunohistochemistry, immunofluorescence, ELISA assays,radioimmunoassay, FACS analysis, immunoprecipitation, immunoblotanalysis, Pandex microfluorimetric assay, agglutination assays, flowcytometry and serum diagnostic assays, which are well known in the art(Harlow and Lane, supra, 1988; Harlow and Lane, Using Antibodies: ALaboratory Manual, Cold Spring Harbor Press (1999)).

An antibody can be made detectable by various means well known in theart. For example, a detectable marker can be directly attached to theantibody or indirectly attached using, for example, a secondary agentthat recognizes the CARD specific antibody. Useful markers include, forexample, radionucleotides, enzymes, binding proteins such as biotin,fluorogens, chromogens and chemiluminescent labels.

An antibody can also be detectable by, for example, a fluorescentlabeling agent that chemically binds to antibodies or antigens withoutdenaturation to form a fluorochrome (dye) that is a usefulimmunofluorescent tracer. A description of immunofluorescent analytictechniques is found in DeLuca, “Immunofluorescence Analysis”, inAntibody As a Tool, Marchalonis et al., eds., John Wiley & Sons, Ltd.,pp. 189-231 (1982), which is incorporated herein by reference.

In one embodiment, the indicating group is an enzyme, such ashorseradish peroxidase (HRP), glucose oxidase, and the like. In anotherembodiment, radioactive elements are employed labeling agents. Thelinking of a label to a substrate, i.e., labeling of nucleic acidprobes, antibodies, polypeptides, and proteins, is well known in theart. For instance, an invention antibody can be labeled by metabolicincorporation of radiolabeled amino acids provided in the culturemedium. See, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981).Conventional means of protein conjugation or coupling by activatedfunctional groups are particularly applicable. See, for example,Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978),Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.

In addition to detecting the presence of a CARD-containing polypeptide,invention anti-CARD antibodies are contemplated for use herein to alterthe activity of the CARD-containing polypeptide in living animals, inhumans, or in biological tissues or fluids isolated therefrom.Accordingly, compositions comprising a carrier and an amount of anantibody having specificity for CARD-containing polypeptides effectiveto block naturally occurring ligands or other CARD-associatedpolypeptides from binding to invention CARD-containing polypeptides arecontemplated herein.

The present invention further provides transgenic non-human mammals thatare capable of expressing exogenous nucleic acids encodingCARD-containing polypeptides. As employed herein, the phrase “exogenousnucleic acid” refers to nucleic acid sequence which is not native to thehost, or which is present in the host in other than its nativeenvironment, for example, as part of a genetically engineered DNAconstruct. In addition to naturally occurring CARD-containingpolypeptide levels, a CARD-containing polypeptide of the invention caneither be overexpressed or underexpressed in transgenic mammals, forexample, underexpressed in a knock-out animal.

Also provided are transgenic non-human mammals capable of expressingCARD-encoding nucleic acids so mutated as to be incapable of normalactivity. Therefore, the transgenic non-human mammals do not expressnative CARD-containing polypeptide or have reduced expression of nativeCARD-containing polypeptide. The present invention also providestransgenic non-human mammals having a genome comprising antisensenucleic acids complementary to CARD-encoding nucleic acids, placed so asto be transcribed into antisense mRNA complementary to CARD-encodingmRNA, which hybridizes to the mRNA and, thereby, reduces the translationthereof. The nucleic acid can additionally comprise an induciblepromoter and/or tissue specific regulatory elements, so that expressioncan be induced, or restricted to specific cell types.

Animal model systems useful for elucidating the physiological andbehavioral roles of CARD-containing polypeptides are also provided, andare produced by creating transgenic animals in which the expression ofthe CARD-containing polypeptide is altered using a variety oftechniques. Examples of such techniques include the insertion of normalor mutant versions of nucleic acids encoding a CARD-containingpolypeptide by microinjection, retroviral infection or other means wellknown to those skilled in the art, into appropriate fertilized embryosto produce a transgenic animal, see, for example, Hogan et al.,Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring HarborLaboratory, (1986)). Transgenic animal model systems are useful for invivo screening of compounds for identification of specific ligands, suchas agonists or antagonists, which activate or inhibit a biologicalactivity.

Also contemplated herein, is the use of homologous recombination ofmutant or normal versions of CARD-encoding genes with the native genelocus in transgenic animals, to alter the regulation of expression orthe structure of CARD-containing polypeptides by replacing theendogeneous gene with a recombinant or mutated CARD-encoding gene.Methods for producing a transgenic non-human mammal including a geneknock-out non-human mammal, are well known to those skilled in the art(see, Capecchi et al., Science 244:1288 (1989); Zimmer et al., Nature338:150 (1989); Shastry, Experentia, 51:1028-1039 (1995); Shastry, Mol.Cell. Biochem., 181:163-179 (1998); and U.S. Pat. No. 5,616,491, issuedApr. 1, 1997, No. 5,750,826, issued May 12, 1998, and No. 5,981,830,issued Nov. 9, 1999).

In addition to homologous recombination, additional methods such asmicroinjection can be used which add genes to the host genome withoutremoving host genes. Microinjection can produce a transgenic animal thatis capable of expressing both endogenous and exogenous CARD-containingpolypeptides. Inducible promoters can be linked to the coding region ofnucleic acids to provide a means to regulate expression of thetransgene. Tissue specific regulatory elements can be linked to thecoding region to permit tissue-specific expression of the transgene.Transgenic animal model systems are useful for in vivo screening ofcompounds for identification of specific ligands, i.e., agonists andantagonists which activate or inhibit CARD-containing polypeptideresponses.

In accordance with another embodiment of the invention, a method isprovided for identifying a CARD-associated polypeptide (CAP). The methodis carried out by contacting an invention CARD-containing polypeptidewith a candidate CAP and detecting association of the CARD-containingpolypeptide with the CAP.

As used herein, the term “CARD-associated polypeptide” or “CAP” means apolypeptide that can specifically bind to the CARD-containingpolypeptides of the invention, or to any functional fragment of aCARD-containing polypeptide of the invention. Because CARD-containingpolypeptides of the invention contain domains which can self-associate,other CARD-containing polypeptides are exemplary CAPs. Other exemplaryCAPs are proteins and fragments thereof that can bind a CARD, ERM, PDZor filament domain of an invention CARD-containing polypeptide. Forexample, cytoskeletal components that bind the filament domain ofCARD-10X or the ERM domain of CARD-11X are exemplary CAPs.

A CAP can be identified and its binding with a CARD-containingpolypeptide of the invention further characterized, for example, usingin vitro protein binding assays similar to those described in, forexample, Ausubel et al., supra, 2000, including co-immunoprecipitationassays, sedimentation assays, affinity chromatography, gel-overlayassays, radiolabeled ligand binding assays, surface plasmon resonance(SPR) on BIAcore, nuclear magnetic resonance (NMR) spectroscopy,circular dichroism (CD) spectroscopy, and mass spectroscopy. A CAP canalso be identified and characterized in vivo using protein-interactionassays and methods known in the art, including yeast two-hybrid assaysand FRET-based binding assays.

Normal association of CARD-containing polypeptide and a CAP polypeptidein a cell can be altered due, for example, to the expression in the cellof a variant CAP or CARD-containing polypeptide, respectively, either ofwhich can compete with the normal binding function of a CARD-containingpolypeptide and, therefore, can decrease the association of CAP andCARD-containing polypeptides in a cell. The term “variant” is usedgenerally herein to mean a polypeptide that is different from the CAP orCARD-containing polypeptide that normally is found in a particular celltype. Thus, a variant can include a mutated protein or a naturallyoccurring protein, such as an isoform, that is not normally found in aparticular cell type.

As used herein, a “candidate CAP” refers to a polypeptide containing asequence known or suspected of binding one or more CARD-containingpolypeptides of the invention. Thus, a CAP can represent a full-lengthprotein or a CARD-associating fragment thereof. Likewise, a CAP-encodingnucleic acid need not encode the full-length protein, but only theCARD-associating fragment of the CAP.

Since CARD-containing polypeptides can be involved in apoptosis andanoikis, the association of a CAP with a CARD-containing polypeptide canaffect the sensitivity or resistance of a cell to apoptosis or caninduce or block apoptosis induced by external or internal stimuli. Theidentification of various CAPs by use of known methods can be used todetermine the function of these CAPs in cell death or signaltransduction pathways controlled by CARD-containing polypeptides,allowing for the development of assays that are useful for identifyingagents that effectively alter the association of a CAP with aCARD-containing polypeptide. Such agents can be useful for providingeffective therapy for conditions caused, at least in part, byinsufficient apoptosis, such as a cancer, autoimmune disease or certainviral infections. Such agents can also be useful for providing aneffective therapy for diseases where excessive apoptosis is known tooccur, such as stroke, heart failure, or AIDS.

A further embodiment of the invention provides a method to identifyagents that can effectively alter CARD-containing polypeptide activity,for example the ability of CARD-containing polypeptides to associatewith one or more CAPs. Thus, the present invention provides a screeningassay useful for identifying an effective agent, which can alter theassociation of a CARD-containing polypeptide with a CARD-associatedpolypeptide (CAP), such as a heterologous CARD-containing polypeptide.Since CARD-containing polypeptides are involved in biochemical processessuch as apoptosis, the identification of such effective agents can beuseful for altering the level of a biochemical process such as apoptosisin a cell, for example in a cell of a subject having a pathologycharacterized by an increased or decreased level of apoptosis.

Further, effective agents can be useful for alteration of otherbiochemical process modulated by a CARD-containing polypeptide of theinvention, including, for example, NF-κB induction, cytokine processing,cytokine receptor signaling, caspase-mediated proteolysis, cytoskeletalintegrity, inflammation and cell adhesion.

As used herein, the term “agent” means a chemical or biological moleculesuch as a simple or complex organic molecule, a peptide, apeptido-mimetic, a polypeptide, a protein or an oligonucleotide that hasthe potential for altering the association of a CARD-containingpolypeptide with a heterologous protein or altering the ability of aCARD-containing polypeptide to self-associate or altering the ligandbinding or catalytic activity of a CARD-containing polypeptide. Anexemplary ligand binding activity is nucleotide binding activity, suchas ADP or ATP binding activity; and exemplary catalytic activities arenucleotide hydrolytic activity and proteolytic activity. In addition,the term “effective agent” is used herein to mean an agent that isconfirmed as capable of altering the association of a CARD-containingpolypeptide with a heterologous protein or altering the ability of aCARD-containing polypeptide to self-associate or altering the ligandbinding or catalytic activity of a CARD-containing polypeptide. Forexample, an effective agent may be an anti-CARD antibody, aCARD-associated polypeptide, a caspase inhibitor, and the like.

As used herein, the term “alter the association” means that theassociation between two specifically interacting polypeptides either isincreased or decreased due to the presence of an effective agent. As aresult of an altered association of CARD-containing polypeptide withanother polypeptide in a cell, the activity of the CARD-containingpolypeptide or the CAP can be increased or decreased, thereby altering abiochemical process, for example, the level of apoptosis in the cell. Asused herein, the term “alter the activity” means that the agent canincrease or decrease the activity of a CARD-containing polypeptide in acell, thereby modulating a biochemical process in a cell, for example,the level of apoptosis in the cell. Similarly, the term “alter thelevel” of a biological process modulated by a CARD-containingpolypeptide refers to an increase or decrease a biochemical processwhich occurs upon altering the activity of a CARD-containingpolypeptide. For example, an effective agent can increase or decreasethe CARD:CARD-associating activity of a CARD-containing polypeptide,which can result in decreased apoptosis. An effective agent can alsoincrease or decrease the association between the ERM domain, filamentdomain or PDZ domain of a polypeptide of the invention and a cellularcomponent, thereby altering cytoskeletal organization.

An effective agent can act by interfering with the ability of aCARD-containing polypeptide to associate with another polypeptide, orcan act by causing the dissociation of a CARD-containing polypeptidefrom a complex with a CARD-associated polypeptide, wherein the ratio ofbound CARD-containing polypeptide to free CARD-containing polypeptide isrelated to the level of a biochemical process, such as, apoptosis, in acell. For example, binding of a ligand to a CAP can allow the CAP, inturn, to bind a specific CARD-containing polypeptide such that all ofthe specific CARD-containing polypeptide is bound to a CAP, and canresult in decreased apoptosis. The association, for example, of aCARD-containing polypeptide and a CARD-containing polypeptide can resultin activation or inhibition of the NB-ARC:NB-ARC-associating activity ofa CARD-containing polypeptide. In the presence of an effective agent,the association of a CARD-containing polypeptide and a CAP can bealtered, which can, for example, alter the activation of caspases in thecell. As a result of the altered caspase activation, the level ofapoptosis in a cell can be increased or decreased. Thus, theidentification of an effective agent that alters the association of aCARD-containing polypeptide with another polypeptide can allow for theuse of the effective agent to increase or decrease the level of abiological process such as apoptosis.

An effective agent can be useful, for example, to increase the level ofapoptosis in a cell such as a cancer cell, which is characterized byhaving a decreased level of apoptosis as compared to its normal cellcounterpart. An effective agent also can be useful, for example, todecrease the level of apoptosis in a cell such as a T lymphocyte in asubject having a viral disease such as acquired immunodeficiencysyndrome, which is characterized by an increased level of apoptosis inan infected T cell as compared to a normal T cell. Thus, an effectiveagent can be useful as a medicament for altering the level of apoptosisin a subject having a pathology characterized by increased or decreasedapoptosis. In addition, an effective agent can be used, for example, todecrease the level of apoptosis and, therefore, increase the survivaltime of a cell such as a hybridoma cell in culture. The use of aneffective agent to prolong the survival of a cell in vitro cansignificantly improve bioproduction yields in industrial tissue cultureapplications.

It will be appreciated that a functional fragment or peptide of aCARD-containing polypeptide or a CAP can be an effective agent, so longas it alters the association between a CARD-containing polypeptide and aCAP. Such peptides, which can be as small as about five amino acids, canbe identified, for example, by screening a peptide library (see, forexample, Ladner et al., U.S. Pat. No. 5,223,409, which is incorporatedherein by reference) to identify peptides that can bind aCARD-containing polypeptide or a CARD-associated polypeptide.

Such peptide effective agents can act by decreasing the association of aCARD-containing polypeptide with a CAP in a cell by competing forbinding to the CARD-containing polypeptide. A non-naturally occurringpeptido-mimetic also can be useful as an effective agent. Such apeptido-mimetic can include, for example, a peptoid, which ispeptide-like sequence containing N-substituted glycines, or anoligocarbamate. A peptido-mimetic can be particularly useful as aneffective agent due, for example, to having an increased stability toenzymatic degradation in vivo.

In accordance with another embodiment of the present invention, there isprovided a method of identifying an effective agent that alters theassociation of an invention CARD-containing polypeptide with aCARD-associated polypeptide (CAP), by the steps of:

(a) contacting the CARD-containing polypeptide and CAP polypeptide underconditions that allow the polypeptides to associate, with an agentsuspected of being able to alter the association of the CARD-containingpolypeptide and CAP polypeptides; and

(b) determining association of the CARD-containing polypeptide and theCAP polypeptide, where an agent that alters the association isidentified as an effective agent.

Methods are well-known in the art for detecting the altered associationof the CARD-containing polypeptide and CAP polypeptides, for example,measuring protein:protein binding, protein degradation or apoptoticactivity can be employed in bioassays described herein to identifyagents as agonists or antagonists of CARD-containing polypeptides. Asdescribed herein, CARD-containing polypeptides have the ability toself-associate. Thus, methods for identifying effective agents thatalter the association of a CARD-containing polypeptide with a CAP areuseful for identifying effective agents that alter the ability of aCARD-containing polypeptide to self-associate.

As used herein, “conditions that allow a CARD-containing polypeptide anda CAP polypeptide to associate” refers to environmental conditions inwhich a CARD-containing polypeptide and CAP specifically associate. Suchconditions will typically be aqueous conditions, with a pH between 3.0and 11.0, and temperature below 100° C. Preferably, the conditions willbe aqueous conditions with salt concentrations below the equivalent of 1M NaCl, and pH between 5.0 and 9.0, and temperatures between 0° C. and50° C. Most preferably, the conditions will range from physiologicalconditions of normal yeast or mammalian cells, or conditions favorablefor carrying out in vitro assays such as immunoprecipitation and GSTprotein:protein association assays, and the like.

The present invention also provides in vitro screening assays. Suchscreening assays are particularly useful in that they can be automated,which allows for high through-put screening, for example, of randomly orrationally designed agents such as drugs, peptidomimetics or peptides inorder to identify those agents that effectively alter the association ofa CARD-containing polypeptide and a CAP or the catalytic or ligandbinding activity of a CARD-containing polypeptide and, thereby, alter abiochemical process modulated by a CARD-containing polypeptide such asapoptosis. An in vitro screening assay can utilize, for example, aCARD-containing polypeptide including a CARD-containing fusion proteinsuch as a CARD-glutathione-S-transferase fusion protein. For use in thein vitro screening assay, the CARD-containing polypeptide should have anaffinity for a solid substrate as well as the ability to associate witha CARD-associated polypeptide. For example, when a CARD-containingpolypeptide is used in the assay, the solid substrate can contain acovalently attached anti-CARD antibody. Alternatively, a GST/CARD fusionprotein can be used in the assay and the solid substrate can containcovalently attached glutathione, which is bound by the GST component ofthe GST/CARD fusion protein. Similarly, a CARD-associated polypeptidecan be used in such screening assays.

An in vitro screening assay can be performed, for example, by allowing aCARD-containing polypeptide to bind to the solid support, then adding aCARD-associated polypeptide and an agent to be tested. Referencereactions, which do not contain an agent, can be performed in parallel.Following incubation under suitable conditions, which include, forexample, an appropriate buffer concentration and pH and time andtemperature that permit binding of the particular CARD-containingpolypeptide and CARD-associated polypeptide, the amount of protein thathas associated in the absence of an agent and in the presence of anagent can be determined. The association of a CARD-associatedpolypeptide with a CARD-containing polypeptide can be detected, forexample, by attaching a detectable moiety such as a radionuclide or afluorescent label to a CARD-associated polypeptide and measuring theamount of label that is associated with the solid support, wherein theamount of label detected indicates the amount of association of theCARD-associated polypeptide with a CARD-containing polypeptide. Aneffective agent is determined by comparing the amount of specificbinding in the presence of an agent as compared to a reference level ofbinding, wherein an effective agent alters the association ofCARD-containing polypeptide with the CARD-associated polypeptide. Suchan assay is particularly useful for screening a panel of agents such asa peptide library in order to detect an effective agent.

Various binding assays to identify cellular proteins that interact withprotein binding domains are known in the art and include, for example,yeast two-hybrid screening assays (see, for example, U.S. Pat. Nos.5,283,173, 5,468,614 and 5,667,973; Ausubel et al., supra, 2000; Lubanet al., Curr. Opin. Biotechnol. 6:59-64 (1995)) and affinity columnchromatography methods using cellular extracts. By synthesizing orexpressing polypeptide fragments containing various CARD-associatingsequences or deletions, the CARD binding interface can be readilyidentified.

Another assay for screening of agents that alter the activity of aCARD-containing polypeptide is based on altering the phenotype of yeastby expressing a CARD-containing polypeptide. In one embodiment,expression of a CARD-containing polypeptide can be inducible (Tao etal., J. Biol. Chem. 273:23704-23708 (1998), and the compounds can bescreened when CARD-containing polypeptide expression is induced.CARD-containing polypeptides of the invention can also be co-expressedin yeast with CAP polypeptides used to screen for compounds thatantagonize the activity of the CARD-containing polypeptide.

Also provided by the present invention are assays to identify agentsthat alter CARD-containing polypeptide expression. Methods to determineCARD-containing polypeptide expression can involve detecting a change inCARD-containing polypeptide abundance in response to contacting the cellwith an agent that modulates CARD-containing polypeptide expression.Assays for detecting changes in polypeptide expression include, forexample, immunoassays with CARD-specific antibodies, such asimmunoblotting, immunofluorescence, immunohistochemistry andimmunoprecipitation assays, as described herein.

As understood by those of skill in the art, assay methods foridentifying agents that alter. CARD-containing polypeptide activitygenerally require comparison to a reference. One type of a “reference”is a cell or culture that is treated substantially the same as the testcell or test culture exposed to the agent, with the distinction that the“reference” cell or culture is not exposed to the agent. Another type of“reference” cell or culture can be a cell or culture that is identicalto the test cells, with the exception that the “reference” cells orculture do not express a CARD-containing polypeptide. Accordingly, theresponse of the transfected cell to an agent is compared to theresponse, or lack thereof, of the “reference” cell or culture to thesame agent under the same reaction conditions.

Methods for producing pluralities of agents to use in screening forcompounds that alter the activity of a CARD-containing polypeptide,including chemical or biological molecules such as simple or complexorganic molecules, metal-containing compounds, carbohydrates, peptides,proteins, peptidomimetics, glycoproteins, lipoproteins, nucleic acids,antibodies, and the like, are well known in the art and are described,for example, in Huse, U.S. Pat. No. 5,264,563; Francis et al., Curr.Opin. Chem. Biol. 2:422-428 (1998); Tietze et al., Curr. Biol.,2:363-371 (1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al.,Med. Res. Rev. 15:481-496 (1995); and the like. Libraries containinglarge numbers of natural and synthetic agents also can be obtained fromcommercial sources. Combinatorial libraries of molecules can be preparedusing well known combinatorial chemistry methods (Gordon et al., J. Med.Chem. 37: 1233-1251 (1994); Gordon et al., J. Med. Chem. 37: 1385-1401(1994); Gordon et al., Acc. Chem. Res. 29:144-154 (1996); Wilson andCzarnik, eds., Combinatorial Chemistry: Synthesis and Application, JohnWiley & Sons, New York (1997)).

The invention further provides a method of diagnosing or predictingclinical prognosis of a pathology characterized by an increased ordecreased level of a CARD-containing polypeptide in a subject. Themethod includes the steps of (a) obtaining a test sample from thesubject; (b) contacting the sample with an agent that can bind aCARD-containing polypeptide or nucleic acid molecule of the inventionunder suitable conditions, wherein the conditions allow specific bindingof the agent to the CARD-containing polypeptide; and (c) comparing theamount of the specific binding in the test sample with the amount ofspecific binding in a reference sample, wherein an increased ordecreased amount of the specific binding in the test sample as comparedto the reference sample is diagnostic of, or predictive of the clinicalprognosis of, a pathology. The agent can be, for example, an anti-CARDantibody, a CARD-associated-polypeptide (CAP), or a CARD-encodingnucleic acid.

Exemplary pathologies for diagnosis or the prediction of clinicalprognosis include any of the pathologies described herein, such asneoplastic pathologies (e.g. cancer), autoimmune diseases, and otherpathologies related to abnormal cell proliferation or abnormal celldeath (e.g. apoptosis), as disclosed herein.

The invention also provides a method of diagnosing cancer or monitoringcancer therapy by contacting a test sample from a patient with aCARD-specific antibody. The invention additionally provides a method ofassessing prognosis (e.g., predicting the clinical prognosis) ofpatients with cancer comprising contacting a test sample from a patientwith a CARD-specific antibody.

The invention additionally provides a method of diagnosing cancer ormonitoring cancer therapy by contacting a test sample from a patientwith a oligonucleotide that selectively hybridizes to a CARD-encodingnucleic acid molecule. The invention further provides a method ofassessing prognosis (e.g., predicting the clinical prognosis) ofpatients with cancer by contacting a test sample from a patient with aoligonucleotide that selectively hybridizes to a CARD-encoding nucleicacid molecule.

The methods of the invention for diagnosing cancer or monitoring cancertherapy using a CARD-specific antibody or oligonucleotide or nucleicacid that selectively hybridizes to a CARD-encoding nucleic acidmolecule can be used, for example, to segregate patients into a highrisk group or a low risk group for diagnosing cancer or predicting riskof metastasis or risk of failure to respond to therapy. Therefore, themethods of the invention can be advantageously used to determine, forexample, the risk of metastasis in a cancer patient, or the risk of anautoimmune disease of a patient, or as a prognostic indicator ofsurvival or disease progression in a cancer patient or patient with anautoimmune disease. One of ordinary skill in the art would appreciatethat the prognostic indicators of survival for cancer patients sufferingfrom stage I cancer can be different from those for cancer patientssuffering from stage IV cancer. For example, prognosis for stage Icancer patients can be oriented toward the likelihood of continuedgrowth and/or metastasis of the cancer, whereas prognosis for stage IVcancer patients can be oriented toward the likely effectiveness oftherapeutic methods for treating the cancer. Accordingly, the methods ofthe invention directed to measuring the level of or determining thepresence of a CARD-containing polypeptide or CARD-encoding nucleic acidcan be used advantageously as a prognostic indicator for the presence orprogression of a cancer or response to therapy.

The present invention also provides therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention, such as pharmaceuticalcompositions, contain a physiologically compatible carrier together withan invention CARD-containing polypeptide (or functional fragmentthereof), an invention CARD-encoding nucleic acid, an agent that altersCARD activity or expression identified by the methods described herein,or an anti-CARD antibody, as described herein, dissolved or dispersedtherein as an active ingredient. In a preferred embodiment, thetherapeutic composition is not immunogenic when administered to a mammalor human patient for therapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically compatible” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset, and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well known in the art.Typically such compositions are prepared as injectables either as liquidsolutions or suspensions; however, solid forms suitable for solution, orsuspension, in liquid prior to use can also be prepared. The preparationcan also be emulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient inamounts suitable for use in the therapeutic methods described herein.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like, as well as combinations of any two or morethereof. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents, and the like, which enhance the effectiveness ofthe active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable nontoxic salts include the acid additionsalts (formed with the free amino groups of the polypeptide) that areformed with inorganic acids such as, for example, hydrochloric acid,hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolicacid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinicacid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid,naphthalene sulfonic acid, sulfanilic acid, and the like.

Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and the like; and organic bases such asmono-, di-, and tri-alkyl and -aryl amines (e.g., triethylamine,diisopropyl amine, methyl amine, dimethyl amine, and the like) andoptionally substituted ethanolamines (e.g., ethanolamine,diethanolamine, and the like).

Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH, physiological saline orboth, such as phosphate-buffered saline. Still further, aqueous carrierscan contain more than one buffer salt, as well as salts such as sodiumand potassium chlorides, dextrose, polyethylene glycol and othersolutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary additional liquid phases includeglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

As described herein, an “effective amount” is a predetermined amountcalculated to achieve the desired therapeutic effect, i.e., to alter theprotein binding activity of a CARD-containing polypeptide or otherbiological activity, resulting in altered biochemical process modulatedby a CARD-containing polypeptide. The required dosage will vary with theparticular treatment and with the duration of desired treatment;however, it is anticipated that dosages between about 10 micrograms andabout 1 milligram per kilogram of body weight per day will be used fortherapeutic treatment. It may be particularly advantageous to administersuch agents in depot or long-lasting form as discussed herein. Atherapeutically effective amount is typically an amount of an agentidentified herein that, when administered in a physiologicallyacceptable composition, is sufficient to achieve a plasma concentrationof from about 0.1 μg/ml to about 100 μg/ml, preferably from about 1.0μg/ml to about 50 μg/ml, more preferably at least about 2 μg/ml andusually 5 to 10 μg/ml. Therapeutic invention anti-CARD antibodies can beadministered in proportionately appropriate amounts in accordance withknown practices in this art.

Also provided herein are methods of treating pathologies characterizedby abnormal cell proliferation, abnormal cell death, or inflammationsaid method comprising administering an effective amount of an inventiontherapeutic composition. Such compositions are typically administered ina physiologically compatible composition.

Exemplary abnormal cell proliferation diseases associated withCARD-containing polypeptides contemplated herein for treatment accordingto the present invention include cancer pathologies, keratinocytehyperplasia, neoplasia, keloid, benign prostatic hypertrophy,inflammatory hyperplasia, fibrosis, smooth muscle cell proliferation inarteries following balloon angioplasty (restenosis), and the like.Exemplary cancer pathologies contemplated herein for treatment include,gliomas, carcinomas, adenocarcinomas, sarcomas, melanomas, hamartomas,leukemias, lymphomas, and the like. Further diseases associated withCARD-containing polypeptides contemplated herein for treatment accordingto the present invention include inflammatory diseases and diseases ofcell loss. Such diseases include allergies, inflammatory diseasesincluding arthritis, lupus, Schrogen's syndrome, Crohn's disease,ulcerative colitis, as well as allograft rejection, such asgraft-versus-host disease, and the like. CARD-containing polypeptidescan also be useful in design of strategies for preventing diseasesrelated to abnormal cell death in conditions such as stroke, myocardialinfarction, heart failure, neurodegenerative diseases such asParkinson's and Alzheimer's diseases, and for immunodeficiencyassociated diseases such as HIV infection, HIV-related disease, and thelike.

Methods of treating pathologies can include methods of modulating theactivity of one or more oncogenic proteins, wherein the oncogenicproteins specifically interact with a CARD-containing polypeptide of theinvention. Methods of modulating the activity of such oncogenic proteinswill include contacting the oncogenic protein with a substantially pureCARD-containing polypeptide or an active fragment (i.e., oncogenicprotein-binding fragment) thereof. This contacting will alter theactivity of the oncogenic protein, thereby providing a method oftreating a pathology caused by the oncogenic protein. Further methods ofmodulating the activity of oncogenic proteins will include contactingthe oncogenic protein with an agent, wherein the agent altersinteraction between a CARD-containing polypeptide and an oncogenicprotein.

Also contemplated herein, are therapeutic methods using inventionpharmaceutical compositions for the treatment of pathological disordersin which there is too little cell division, such as, for example, bonemarrow aplasias, immunodeficiencies due to a decreased number oflymphocytes, and the like. Methods of treating a variety of inflammatorydiseases with invention therapeutic compositions are also contemplatedherein, such as treatment of sepsis, fibrosis (e.g., scarring),arthritis, graft versus host disease, and the like.

The present invention also provides methods for diagnosing a pathologythat is characterized by an increased or decreased level of abiochemical process to determine whether the increased or decreasedlevel of the biochemical process is due, for example, to increased ordecreased expression of a CARD-containing polypeptide or to expressionof a variant CARD-containing polypeptide. As disclosed herein, suchbiochemical processes include apoptosis, NF-κB induction, cytokineprocessing, caspase-mediated proteolysis, transcription, inflammation,cell adhesion, cytoskeletal integrity and the like.

The identification of such a pathology can allow for interventiontherapy using an effective agent, nucleic acid molecule, antisenseoligonucleotide or polypeptide as described herein. In general, a testsample can be obtained from a subject having a pathology characterizedby having or suspected of having increased or decreased apoptosis andcan be compared to a reference sample from a normal subject to determinewhether a cell in the test sample has, for example, increased ordecreased expression of a CARD-encoding gene. The level of aCARD-containing polypeptide in a cell can be determined by contacting asample with a reagent such as an anti-CARD antibody or a CARD-associatedpolypeptide, either of which can specifically bind a CARD-containingpolypeptide. For example, the level of a CARD-containing polypeptide ina cell can determined by well known immunoassay or immunohistochemicalmethods using an anti-CARD antibody (see, for example, Reed et al.,Anal. Biochem. 205:70-76 (1992); see, also, Harlow and Lane, supra,(1988)).

As used herein, the term “reagent” means a chemical or biologicalmolecule that can specifically bind to a CARD-containing polypeptide orto a bound CARD/CARD-associated polypeptide complex. For example, eitheran anti-CARD antibody or a CARD-associated polypeptide can be a reagentfor a CARD-containing polypeptide, whereas either an anti-CARD antibodyor an anti-CARD-associated polypeptide antibody can be a reagent for aCARD/CARD-associated polypeptide complex.

Increased or decreased expression of a CARD-encoding gene in a cell in atest sample can be determined, for example, by comparison to an expectednormal level of CARD-containing polypeptide or CARD-encoding mRNA in aparticular cell type. A normal range of CARD-containing polypeptide orCARD-encoding mRNA levels in various cell types can be determined bysampling a statistically significant number of normal subjects. Inaddition, a reference sample can be evaluated in parallel with a testsample in order to determine whether a pathology characterized byincreased or decreased apoptosis is due to increased or decreasedexpression of a CARD-encoding gene. The test sample can be examinedusing, for example, immunohistochemical methods as described above orthe sample can be further processed and examined. For example, anextract of a test sample can be prepared and examined to determinewhether a CARD-containing polypeptide in the sample can associate with aCARD-associated polypeptide in the same manner as a CARD-containingpolypeptide from a reference cell or whether, instead, a variantCARD-containing polypeptide is expressed in the cell.

In accordance with another embodiment of the present invention, thereare provided diagnostic systems, preferably in kit form, comprising atleast one invention CARD-encoding nucleic acid, CARD-containingpolypeptide, and/or anti-CARD antibody described herein, in a suitablepackaging material. In one embodiment, for example, the diagnosticnucleic acids are derived from any of SEQ ID NOS:1, 7 or 15. Inventiondiagnostic systems are useful for assaying for the presence or absenceof CARD-encoding nucleic acid in either genomic DNA or in transcribedCARD-encoding nucleic acid, such as mRNA or cDNA.

A suitable diagnostic system includes at least one inventionCARD-encoding nucleic acid, CARD-containing polypeptide, and/oranti-CARD antibody, preferably two or more invention nucleic acids,proteins and/or antibodies, as a separately packaged chemical reagent(s)in an amount sufficient for at least one assay. Instructions for use ofthe packaged reagent are also typically included. Those of skill in theart can readily incorporate invention nucleic acid probes and/or primersinto kit form in combination with appropriate buffers and solutions forthe practice of the invention methods as described herein.

As employed herein, the phrase “packaging material” refers to one ormore physical structures used to house the contents of the kit, such asinvention nucleic acid probes or primers, and the like. The packagingmaterial is constructed by well known methods, preferably to provide asterile, contaminant-free environment. The packaging material has alabel which indicates that the invention nucleic acids can be used fordetecting a particular CARD-encoding sequence including the nucleotidesequences set forth in SEQ ID NOS:1, 7 or 15 or mutations or deletionstherein, thereby diagnosing the presence of, or a predisposition for apathology such as cancer or an autoimmune disease. In addition, thepackaging material contains instructions indicating how the materialswithin the kit are employed both to detect a particular sequence anddiagnose the presence of, or a predisposition for a pathology such ascancer or an autoimmune disease.

The packaging materials employed herein in relation to diagnosticsystems are those customarily utilized in nucleic acid-based diagnosticsystems. As used herein, the term “package” refers to a solid matrix ormaterial such as glass, plastic, paper, foil, and the like, capable ofholding within fixed limits an isolated nucleic acid, oligonucleotide,or primer of the present invention. Thus, for example, a package can bea glass vial used to contain milligram quantities of a contemplatednucleic acid, oligonucleotide or primer, or it can be a microtiter platewell to which microgram quantities of a contemplated nucleic acid probehave been operatively affixed.

“Instructions for use” typically include a tangible expressiondescribing the reagent concentration or at least one assay methodparameter, such as the relative amounts of reagent and sample to beadmixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions, and the like.

A diagnostic assay should include a simple method for detecting theamount of a CARD-containing polypeptide or CARD-encoding nucleic acid ina sample that is bound to the reagent. Detection can be performed bylabeling the reagent and detecting the presence of the label using wellknown methods (see, for example, Harlow and Lane, supra, 1988; chap. 9,for labeling an antibody). A reagent can be labeled with variousdetectable moieties including a radiolabel, an enzyme, biotin or afluorochrome. Materials for labeling the reagent can be included in thediagnostic kit or can be purchased separately from a commercial source.Following contact of a labeled reagent with a test sample and, ifdesired, a control sample, specifically bound reagent can be identifiedby detecting the particular moiety.

A labeled antibody that can specifically bind the reagent also can beused to identify specific binding of an unlabeled reagent. For example,if the reagent is an anti-CARD antibody, a second antibody can be usedto detect specific binding of the anti-CARD antibody. A second antibodygenerally will be specific for the particular class of the firstantibody. For example, if an anti-CARD antibody is of the IgG class, asecond antibody will be an anti-IgG antibody. Such second antibodies arereadily available from commercial sources. The second antibody can belabeled using a detectable moiety as described above. When a sample islabeled using a second antibody, the sample is first contacted with afirst antibody, then the sample is contacted with the labeled secondantibody, which specifically binds to the first antibody and results ina labeled sample.

In accordance with another embodiment of the invention, there areprovided methods for determining a prognosis of disease free or overallsurvival in a patient suffering from cancer. For example, it iscontemplated herein that abnormal levels of CARD-containing polypeptides(either higher or lower) in primary tumor tissue show a high correlationwith either increased or decreased tumor recurrence or spread, andtherefore indicates the likelihood of disease free or overall survival.Thus, the present invention advantageously provides a significantadvancement in cancer management because early identification ofpatients at risk for tumor recurrence or spread will permit aggressiveearly treatment with significantly enhanced potential for survival. Alsoprovided are methods for predicting the risk of tumor recurrence orspread in an individual having a cancer tumor; methods for screening acancer patient to determine the risk of tumor metastasis; and methodsfor determining the proper course of treatment for a patient sufferingfrom cancer. These methods are carried out by collecting a sample from apatient and comparing the level of CARD-encoding gene expression in thepatient to the level of expression in a control or to a reference levelof CARD-encoding gene expression as defined by patient populationsampling, tissue culture analysis, or any other method known fordetermining reference levels for determination of disease prognosis. Thelevel of CARD-encoding gene expression in the patient is then classifiedas higher than the reference level or lower than the reference level,wherein the prognosis of survival or tumor recurrence is different forpatients with higher levels than the prognosis for patients with lowerlevels.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Identification of CARD-Containing Polypeptides

CARD-10X, -11X and -12X proteins were identified using Saturated Blastsearches (Li et al., “Saturated BLAST: An automated multipleintermediate sequence search used to detect distant homology,”Bioinformatics (2000), in press). A representative set of CARD domainswas used as queries and a cascade of TBLASTN and PSI-BLAST searches wasperformed on nucleotide databases at NCBI (htgs, gss, dbest) and the NRprotein database.

The new candidate CARD-domains were confirmed by:

1) Determining whether the identified nucleotide sequence falls withinan exon as predicted by the GENSCAN program.

2) Identifying EST sequences corresponding to the novel CARD domain.

3) Performing a FFAS fold prediction calculation against a database ofproteins of known structures (PDB) enriched in apoptotic domains(Rychlewski et al., Protein Science 9:232-241 (2000)).

4) Performing a PSI-BLAST search against the NR protein database.

The novelty of the discovered CARD domain was assessed by:

1) Performing a PSI-BLAST search against the NR protein database atNCBI.

2) Performing a PSI-BLAST search against a database of known CARDdomains.

3) Performing a BLASTN search (using the nucleotide sequences ofCARD-10X, 11X, 12X) against the NR nucleotide database at NCBI.

Additional domains in CARD-10X, 11X, 12X (e.g. PDZ, domain, filamentdomain) were identified by performing an exon prediction (GENSCAN)analysis for full genomic contigs in which the CARD domains were found.

The protein sequences obtained in this way were used as queries for FFASsearches against the PDB, PFAM and COG databases, as well as for HMMsearches in the PFAM database (Bateman et al., Nucleic Acids Res.27:260-262 (1999)).

All journal article, reference and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference in their entirety.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

1. An isolated nucleic acid molecule encoding a CARD-containingpolypeptide, wherein said nucleic acid molecule is selected from thegroup consisting of: (a) a nucleic acid molecule encoding a polypeptidecomprising the amino acid sequence of CARD-11X (SEQ ID NO:8) or CARD-12X(SEQ ID NO:16); (b) a nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:7 or SEQ ID NO:15; and (c) a nucleic acid moleculethat hybridizes to the nucleic acid molecule of (a) or (b) undermoderately stringent hybridization conditions.
 2. An isolated nucleicacid molecule encoding a functional fragment of a CARD-containingpolypeptide, wherein said nucleic acid molecule is selected from thegroup consisting of: (a) a nucleic acid molecule encoding the CARDdomain of CARD-10X (SEQ ID NO:4), the filament domain of CARD-10X (SEQID NO:6), the CARD domain of CARD-11X (SEQ ID NO:10), the ERM (ezrin)domain of CARD-11X (SEQ ID NO:12), the PDZ domain of CARD-11X (SEQ IDNO:14) and the CARD domain of CARD-12X (SEQ ID NO:16); (b) a nucleicacid molecule comprising the nucleotide sequence of the CARD domain ofCARD-10X (SEQ ID NO:3), the filament domain of CARD-10X (SEQ ID NO:5),the CARD domain of CARD-11X (SEQ ID NO:9), the ERM (ezrin) domain ofCARD-11X (SEQ ID NO:11), the PDZ domain of CARD-11X (SEQ ID NO:13) andthe CARD domain of CARD-12X (SEQ ID NO:15); and (c) a nucleic acidmolecule that hybridizes to the nucleic acid molecule of (a) or (b)under moderately stringent hybridization conditions, wherein saidnucleic acid molecule does not consist of the nucleotide sequence setforth as SEQ ID NOS:19 or 21-37.
 3. A nucleic acid molecule comprisingsubstantially the same nucleotide sequence as SEQ ID NO:7 or SEQ IDNO:15.
 4. The nucleic acid molecule of claim 1, wherein said nucleicacid molecule is cDNA or mRNA.
 5. A vector containing the nucleic acidmolecule of claim
 1. 6. A cell containing the nucleic acid molecule ofclaim
 1. 7. A composition comprising an amount of the nucleic acidmolecule according to claim 1(c) effective to inhibit expression of aCARD-containing polypeptide, and an acceptable hydrophobic carriercapable of passing through a cell membrane.
 8. An oligonucleotidecomprising at least 15 contiguous nucleotides of the nucleotide sequenceset forth in any of SEQ ID NOS:1, 7 or 15, or the complement thereof,wherein said nucleic acid molecule does not consist of the nucleotidesequence set forth as SEQ ID NOS:19 or 21-37.
 9. The oligonucleotide ofclaim 8, wherein said oligonucleotide is labeled with a detectablemarker.
 10. A kit for detecting the presence of a nucleic acid moleculeencoding a CARD-containing polypeptide, comprising at least oneoligonucleotide according to claim
 9. 11. A substantially purifiedCARD-containing polypeptide, comprising substantially the same aminoacid sequence as the amino acid sequence of CARD-11X (SEQ ID NO:8) orCARD-12X (SEQ ID NO:16).
 12. A substantially purified functionalfragment of a CARD-containing polypeptide, comprising substantially thesame amino acid sequence as the amino acid sequence of the CARD domainof CARD-10X (SEQ ID NO:4), the filament domain of CARD-10X (SEQ IDNO:6), the CARD domain of CARD-11X (SEQ ID NO:10), the ERM (ezrin)domain of CARD-11X (SEQ ID NO:12), the PDZ domain of CARD-11X (SEQ IDNO:14) and the CARD domain of CARD-12X (SEQ ID NO:16), wherein saidfunctional fragment is not encoded by a nucleic acid molecule consistingof the nucleotide sequence set forth as SEQ ID NOS:19 or 21-37. 13.(canceled)
 14. A method of producing a CARD-containing polypeptide,comprising expressing the cDNA of claim 3 in vitro or in a cell underconditions suitable for expression of said polypeptide.
 15. An isolatedanti-CARD antibody having specific reactivity with the CARD-containingpolypeptide of claim
 11. 16. The antibody of claim 15, wherein saidantibody is a monoclonal antibody.
 17. A cell line producing themonoclonal antibody of claim
 16. 18. The antibody of claim 15, whereinsaid antibody is a polyclonal antibody.
 19. A transgenic nonhuman mammalexpressing exogenously the nucleic acid of claim
 1. 20. The transgenicnonhuman mammal of claim 19, wherein said mammal is a mouse. 21-28.(canceled)